BC 21D lab manual2009_10edit.pdf

8/22/2019 BC 21D lab manual2009_10edit.pdf

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University of the West Indies

Mona, Kingston 7

Department of Basic Medical Sciences(Biochemistry)

LABORATORY MANUALBC 21D

2011/20012


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C O N T E N T S

Semester 1

Timetable . 3

Syllabus . 5

Practicals ............................... 6

Accuracy in the laboratory . 11

Chromatographic Techniques .... 23

Protein I ............................... 25

Yeast Invertase ................................... 28

Lysozyme Purification by ion-exchange .......... 31

Banana Polyphenoloxidase (Measuring Browning) . 34

Polyacrylamide Gel Electrophoresis (PAGE) ... 36

Respiratory Control & ATP Synthesis in Mitochondria . 39

Affinity Chromatography of LDH I & II ... 42

Assay of Enzymes Activity 48

Chlorophyll and Photosynthesis . 51


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Syllabus: BC21D

Basic mammalian and plant physiology. Mitochondrial and chloroplast ultrastructure

Biochemical bonding and thermal stability of macromolecules and membranes.Mitochondrial acetyl-CoA formation and utilization. The TCA cycle and the

glyoxylate pathway. The major biosynthetic, intermediary and degradative pathways.

Nitrogen fixation. Bioenergetics as it relates to metabolism. Redox reactions and the

mitochondrial electron transport chain; the chemiosmotic mechanism; oxygenic and

anoxygenic photosynthesis. Special topic(s) in carbohydrate metabolism The

bioenergetics of photosynthetic reactions and of the chemoautotrophs. Transport

across membranes; the mechanisms and bioenergetcs. Induction and repression;

auxotrophic mutants and the elucidation of metabolic pathways.


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PRACTICALS

Introduction

The aims of this practical course are: (i) to introduce common biochemical techniques and

(ii) to supplement and consolidate the contents of lectures. To this end, each student isexpected to perform all of the experiments, and to provide a satisfactory report of each

experiment. It must be emphasized that the lectures and practicals are part of an integral

course, and both must be completed satisfactorily.

The Biochemistry Department has one teaching laboratory which must cater for three

courses taught by the Department; we must ask for your co-operation in keeping the

laboratory running smoothly so that everybody can use the facilities and resources available.This means that all equipment failure must be reported to the supervisor of the practical. All

of you will be familiar with the problems associated with obtaining spare parts from abroad,

so the sooner we know of a fault, the sooner it will be repaired.

There are two main approaches to practical sessions: the more you put in, the more you get

out, or it is an obstacle course which can be negotiated by following instructions. The first

approach is the one we all hope you will adopt.

Before attending the practical session, you must know exactly what you will be trying to

achieve and how you will get about it. You must therefore read up the experiment and plan

the operations to be performed so that they will fit into the precious few hours that yourtimetable will allow you in your laboratory. Some of the experiments are longer than others;

some require several to be going on at once; some cannot be completed in one day. It is part

of your training as a scientist that you know how to plan ahead and work efficiently. Tryand avoid standing around one piece of apparatus waiting for it to become available. Often

you can get another section of the practical going or do some writing up.

Ideally, each experiment should be performed by all students. However, there are

constraints on both your time and our facilities. Therefore, we would like you to work in

pairs or occasionally in larger groups, and to organize the various operations amongst

yourselves so that they can be completed on time.

The supervisor is a member of the academic staff and has overall responsibility for the

session and for assessing you - this means estimating your general competence and marking

your reports. The technical staffs are there to prepare reagents and equipment for thesession. Please try to help them keep the lab running smoothly for all who use it. This

means that you should leave the lab as clean and tidy as possible: wash and clean as much

glassware as you can. Some will require more thorough cleaning, and this the technical staffwill do. Please rinse these things out with water before placing in the baskets and perhaps

more importantly, make sure that any dangerous or unpleasant reagents, eg. strong acids,

etc. have been removed - you know which piece of glassware was used for what, they don't.The demonstrators are there to help you with the experiments and have done many of them

before themselves. If you do not understand anything in the practical, please ask either a

demonstrator or the supervisor.


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Writing up

Practical session reports must be handed in on loose-leaf on the same day the experiment iscompleted. Students who do not hand in work on time will record zero marks for that

particular practical. Retain your reports after marking - we may request them later.

The writing up of your experiment is as important as the practical work. It is essential todevelop a style of writing and a form of presentation which is both clear and concise. In

general scientific journals adhere to a classical form of presentation of experimental papers.

This consists of a division of the material into the following sections: Introduction,Methods, Results, Discussion and Conclusions. In this course, it is not necessary to write

either an introduction or the experimental method since these appear in this practical book.

However, if the method you followed differed from that in the book, you should mention it.

Reports should have the following sections:

a.

Name

b. BC 200c. Title of Experimental and Dated. Objectives of experiment - two or three sentences should

suffice

e. Introduction of experimentf. Results. The results section should contain all the observations and measurements

you made.

Numerical data should be recorded in tables, the boundaries and columns of which

hold be clearly marked. The table headings should be labelled and the units of thequantities always given. All the numbers in a column of a table should be given to

the same number of significant figures unless there is a particular reason for not

doing so. Make sure that the numbers of significant figures reflects the accuracy ofthe technique used for measuring.

Graphs should be drawn whenever appropriate (PROVIDE YOUR OWN PAPER!)

but you should also present the data in tabular form. The axes of graphs should bedrawn in ink and labelled clearly with the parameters plotted and their units stated.

If more than one line is drawn on a graph, then different symbols should be used for

each line. There are unbiased mathematical methods for determining the best line

through a series of experimental points. However, it will be sufficient for you tojudge it visually remembering that (a) the best line may not be straight and (b) may

not pass through any of the points.

g. Discussion. The discussion should be regarded as the core of the write-up.

Here your results should be critically evaluated and conclusions drawn discussed.

Your arguments must be clear and to the point.It is advisable to put sub-headings in your results and discussion sections to assist in

the clarity of presentation.


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Problems

Along with the report, you should also hand in answers to the questions or problems whichare set. These will help to assess your understanding.

Instruments

Some of the equipment you will use is sophisticated and delicate, some of it potentially

lethal. Misuse may cause damage to you or the equipment - we would rather that neitherhappened. Short notes on instrumentation follow.

Centrifuges

Centrifugation is used to increase the rate of sedimentation of particles from a suspension.

There are three types of centrifuges in the Biochemistry Department: bench centrifuges,preparative centrifuges and ultracentrifuges. Undergraduates in the department routinely

use bench centrifuges. The other two types are to be used only by staff and postgraduates.

This is because they are apt to go wrong at the slightest excuse and consequently require

expertise to operate. Bench centrifuges are simple to separate but can be very dangerous.They consist of an electric motor mounted vertically and the drive shaft passes up into the

motor chamber. The speed of the motor is controlled by a variable resistor. The rotor

chamber lid is also part of the protection - should you hear ominous noises from within thechamber DO NOT OPEN THE LID TO LOOK - the opening is at face level and pieces of

moving glass and metal often come shooting out!

Principles of Operation

The drive shaft is mounted vertically and is supported by bearings. If the load being driven(that is the rotor and samples) is not accurately balanced, the bearings will take the train and

wear out. The amount of damage caused to the bearings depends on how unbalanced the

load is and how many times the shaft rotates in this state. In cases of severe imbalance, thewhole centrifuge will jump up and down and move along (or off) the bench.

You must therefore check that the whole load on the drive shaft is balanced. The load will

usually consist of the rotor (sometimes called the centrifuge head) - this can be assumed tobe balanced; metal buckets - these come in different sizes to fit different rotors and can

accommodate centrifuge tubes of different sizes - these are not necessarily of the same

weight. Trunnions - these are to support the buckets in the rotor. Rubber cushions - these

are often ignored to one's peril! Rubber cushions belong in the bottom of the metal bucketsand their function is to cushion the glass centrifuge tube. Finally, we come to the sample

which is invariably contained in a centrifuge tube - test tubes may survive or they may not.

Don't risk it, do the job properly. If you only need one centrifuge tube for your sample, youmust also have a centrifuge tube with water in it to balance the load.

Normal practice is to place buckets, trunnions, cushions and samples on opposite pans of abalance (which must also be checked) and balancing by adjusting the amount of solution in

the samples. The balanced assemblies are then placed diametrically opposite on the rotor,

the lid closed and the rotor accelerated and gently to desired speed. The safe maximum

speed for glass tubes is 3,000 rpm. Higher speeds or too rapid acceleration may shatter the


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tubes. In the event of a tube shattering or any other untoward noise, switch off immediately

and do not open the lid until the speed is aero. Clear away any spillage around or within the

centrifuge. Keep the buckets, trunnions and rubber cushions clean and in the racksprovided, ready for the next user. Do not leave centrifuge running for more than 30-40

minutes or it may overheat.

Spectrophotometry

Many substances of interest in biochemistry absorb light when in solution. A moreconcentrated solution will absorb more light than will a dilute solution. A

spectrophotometer is an instrument which assures the amount of light which passes through

a solution. From this the concentration of the solution can be calculated. Essentially, a

spectrophotometer contains a source of light - a tungsten lamp which gives light in thevisible range of wavelengths, and sometimes a hydrogen or deuterium lamp which gives

light in the ultraviolet range either a prism or a diffraction grating, and a photo-electric cell

which generates an electric current proportional to the intensity of light falling on it.

The Beer-Lambert law governs the absorption of light by substances in spectrophotometers,

and it states that

Log10 = Io = Cl Log10 = Io = ECl

I

The former is the absorbance and when C is expressed in moles/litre, and L in centimeters,

E is the "extinction coefficient" which is characteristic of the solute, for a given wavelength,

solvent and temperature. The spectrophotometer gives

reading of the absorbance or (on a different scale) the percent transmittance (I/Io x 100).

How to use the Spectrophotometer

There will be a variety of spectrophotometers in the Biochemistry Department. You will be

using the Turner instruments in the teaching lab. Consult the information card for detailedinstructions on use. Here are some important points:

a. Allow about 10 minutes for the lamp and instrument to warm up after switching on.

b. The cuvettes must be handled carefully; they are much more expensive than test

tubes.

c. Make sure that there are no greasy finger prints or drops of liquid outside the cuvettewhere they can absorb light and give rinse to incorrect readings. Wipe cuvette after

putting in solution.

d. Adjust the meter so that when no light reaches the photodiode, the meter reads 0%

transmittance ( = infinite absorbance) and when all the incident light reaches the

photodiode (I = Io, because there is no sample), the meter reads 100%transmittance ( = 0 absorbance).

e. Place sample in light path and read absorbance from meter.


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The pH meter

pH meters are frequently used in biochemistry laboratories. The only fault likely to developis a broken electrode and this is always due to ignorance or negligence on the part of the

user. They break because the business end of the electrode has a very thin glass membrane

and is usually either rammed into the bottom of the beaker or smashed by the device stirringthe sample solution. There are, of course, many other ways to destroy these expensiveelectrodes, please contemplate them but DO NOT PRACTISE!!

Principles of Operation

The objective is to measure [H+] in an aqueous solution (pH meters can also measure [H

+] in

organic solvents but this is more complicated and will not concern us here). In fact, it is notH

+] which is measured but [H

+] activity and activity = activity.


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Accuracy in the laboratory

Accuracy may be defined as the degree of conformity to the truth. The scientist therefore

seeks to obtain accurate measurements in the laboratory and in addition to present an

accurate account of the experiment. These aspects of practical work are dealt with in thefirst chapter, since only when these requirements are fulfilled will any practical work be of

value to the student and to those who will read the written report. However, before dealing

with these points, we shall first consider the common units and quantities used inbiochemical experiments.

Units and Quantities

The units used in this book are SI units (Systeme International d'Unites) based on the metric

system. These were approved in 1960 by the General Conference of Weights and Measures

and are being adopted by scientific laboratories throughout the world. They are a coherent

system of units so that if two unit quantities are multiplied or divided, then the answer is theunit of the resultant quantity. In this way the number of multiples and submultiples of units

now in use will be reduced.

Basic units

There are seven units on which all others are based and these are set out in Table 1.1.

Derived units

In addition to those above, there are also a number of derived SI units obtained byappropriate combination of these basic units. For convenience, these derived units are given

special names and

Table 1.1 The SI basic units_________________________________________________________________________

Physical quantity Name Symbol_________________________________________________________________________

Length Metre mMass Kilogramme kgTime Second s

Amount of substance Mole mol

Thermodynamic temperature Kelvin K

Electric current Ampere ALuminous intensity Candela cd

_________________________________________________________________________

those which are likely to be met in biochemical work are listed (Table 1.2).


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Prefixes

Sometimes units may be too large or too small and, in this case, in order to avoid writing toomany zeros, a prefix is placed before the symbol of the unit. The recommended multiples or

fractions of a unit change by 1000 each time (Table 1.3). Thus 0.000 015 mol is written 15

mol and 13 400m is written 13.4km.

The combination of prefix and symbol is regarded as a new symbol so that mm3

means (10-3

m)3

not 10-3

m3. There is therefore no space, point or full stop between the prefix and the

Table 1.2 The special names and symbols for some derived SI units

Definition in terms of units

Name

Physical quantity of unit Symbol Basic Derived

_________________________________________________________________________

Frequency Hertz Hz s-1

-

Force Newton N kg m s-2

J m-1

Energy Joule J kg m2

s-2

N mPressure Pascal Pa kg m

-1s

-2N m

-2

Power Watt W kg m2s

-3J s

-1

Electric charge Coulomb C A s -Electric potential Volt V kg m

2s

-3W A

-1

Electric resistance Ohm kg m2

s-3

A-2

V A-1

Electric capacitance Farad F A2

s4

kg-1

m-2

A s V-1

Customary temperature Degree C Celsius_________________________________________________________________________

None of these units take the plural form so that 5 volts is written 5 V not Vs and 2 metres are

written 2 m not 2 ms.

Table 1.3 Common prefixes for the SI units likely to be used in biochemical work

_________________________________________________________________________

Multiples Fractions

_________________________________________________________________________

Factor Prefix Symbol Factor Prefix Symbol

106

mega M 10-3

milli m10

3kilo k 10

-6micro

10-9

nano n

10-12

pico p_________________________________________________________________________

symbol. However a space is left between symbols in derived units and for the sake of clarity

a point above the line is often used. For example:

ms = millisecond (i.e.) 10-3

s,

whereas

m.s = metre x second (i.e.) m x s


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Units used in conjunction with SI

There are some units used in biochemical work which are unlikely to be replaced altogether

because of their convenience and it is probable that they will continue to be used inconjunction with SI units for some time.

Litre (1). The coherent SI units for volume is, of course, the cubic metre (m3) but this

is rather large and the litre is still accepted as the unit of volume in biochemical work.

The litre is exactly equal to one cubic decimetre (1 decimeter = 10-1

m = dm) so that:

1000 litres = 1 cubic metre = m3

1 litre (1) = 1 dm3

= 10-3

m3

1 millilitre (ml) = 1 cm3

= 10-6

m3

1 microlitre (l) = 1 mm

3

= 10

-9

m

3

The terms millilitre and microlitre will be abandoned in time but probably not until after the

useful life of this text.

Gramme (g). The gramme will continue to be used as an elementary

unit and in association with prefixes (ug, mg) until a new name is adopted for the basic unit

of mass now known as a kilogramme.

Time. The basic SI unit for time is the second but the common units of time (e.g., minute,

hour, year) can still be used when convenient.

Molarity, moles, and concentration

In the experience of the author, difficulty is often encountered by students over moles and

molarity and calculations involving conversions from molarities to millimoles or

micromoles in a given volume. This section should therefore be read and understood beforeattempting any of the experiments involving calculations.

Mole (mol). The basic SI unit of quantity is the mole which gives the amount of a

substance present in say a flask or test tube irrespective of the volume present. It is definedas the molecular weight of a compound in grammes

1 mole = molecular weight in grammes = 6 x 102 3

molecules (Avogdro's number).

The term mole is also applied to other particles of defined composition such as atoms, ions,

or free radicals, as well as molecules:

1 mole of glucose (mol. wt 180) is 180 g.

1 mole of albumin (mol. wt 68 000) is 68 000 g or 68 kg.

Molarity (mol/l). The amount of a substance present in unit volume of solution gives the

concentration of that substance and, in biochemical work, the unit amount is the mole and

the unit volume the litre. A molar solution of a compound is therefore defined as 1 mole of

that compound per litre.


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1 mol/l = mol. wt in grammes per litre of solution

Molarities have been written using M as the symbol (0.15 M - NaCl) but the SI

recommendations is that this be replaced by mol/l (NaCl 0.15 mol/l).

Mass concentration. Sometimes, measurements are made on substances, which do nothave a defined composition, such as the concentration of protein or nucleic acid present in

an extract. In these cases the concentration is expressed in terms of weight per unit volume

rather than moles. It is also used when the molecular weight of the biologically activecompound in a mixture is uncertain, as in the case of vitamin B12 and serum

immunoglobulins. The unit of volume is still the litre, so all concentrations should be

expressed with the litre (g/l, mg/l, g/l, etc.) and not 100 ml as the base. The term % is still

used but should be discontinued, unless clearly defined, because of its ambiguity. Forexample, a 2% solutions of acetic acid could mean:

2 g of acetic acid per 100 g of water (w/w),2 g of acetic acid per 100 ml of water (w/v),

2 ml of acetic acid per 100 ml of water (v/v).

From molarity to moles per millilitre. In many biochemical reactions the number of molesof a substance in the test tube needs to be known, and this can be readily calculated from the

molarity of the solution and the volume present. To do this the number of moles present in

1 ml is first calculated, then multiplied by the volume of the solution present. The followingrelationship is worth remembering and is obtained by decreasing both the amount and

volume by a factor of 103

each time.

A molar solution = 1 mol/l

= 1 mmol/ml= 1 mol/l.

Similarly, a millimolar solution = 1 mmol/l

= 1 mol/ml. Check that you have grasped these ideas bytrying the following calculations, the answers to which are given in the Appendix.

1. How many grammes of glucose are needed to make 100 ml of a molar solution?(Glucose mol. wt = 180.)

2. How many millimoles or micromoles per millilitre are present in the following

solutions: (a) 6 mol/l urea: (b) 0.15 mol/l NaCl; (c) 12 mmol/l fructose; (d) 0.2

mmol/l ATP?

3. How many grammes of glycine are there present in 10 ml of a 20 mmol/l solution?

(Glycine mol. wt = 75.)


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Accurate Measurement

The sources of error

All laboratory work involves some form of measurement and, since all measurements are

liable to errors, the potential sources of these errors should be appreciated.

Human errors. These can arise from a badly designed experiment where insufficient

control is exercised. For example, in many biological experiments the temperature and

illumination of the environment may have profound effects on the effects on the systemunder investigation and should therefore be carefully controlled. Experiments should be

planned in such a way that only one variable is introduced at a time and all other factors that

may affect the experiment are kept constant.

A common source of human error arises from the careless reading of a scale or meniscus

when the problem of parallax is not appreciated. For this reason, many instrument scales

incorporate a mirror behind the pointer so the true reading is obtained when the pointer andits reflection are superimposed. Some manufacturers

Fig. 1.1 Avoiding error due to parallax

overcome this problem by giving a digital readout on their instruments rather than a

deflection on a scale. However, 'digital pipettes' are not yet available and the careless

reading of a meniscus on a pipette is probably one of the greatest sources of error inbiochemical experiments (Fig. 1.1). Human errors such as these can be corrected and, by

careful work, eliminated.

Limitations of apparatus. The limits set by the accuracy of a particular piece of equipment

are usually known and should always be allowed for. For example, the error on a graduated10 ml pipette may be 0.2%, in which case the pipette can deliver a large volume, e.g., 9.2

ml, quite accurately, but the error on trying to deliver 0.1 ml would be as high as 20%.

These errors are also known and can be taken into account.

Standards and blanks. To obtain as accurate a value as possible from an estimation, errors

must be reduced to a minimum and this can be done by careful working and the use ofstandard solutions. Standard solutions of the substance to be estimated should be included

with any test, even when a calibrated instrument and standard reagents are used. This

provides a useful check on the accuracy of a method since the measured figures should fall

within the acceptable limits of the true values. Ideally the standard solution should betreated in an identical manner to the fluid under investigation. A standard curve can then be

constructed showing the variation of the quantity measured with concentration. Values

obtained for the test solution should fall within the range of the standard curve and the value

of the test can then be read. Usually, only one standard is included for volumetricestimations or when a standard curve has previously been constructed.

Blank solutions should be included in any measurements. The same volume of distilledwater replaces the substances to be estimated and the blank is then treated in exactly the

same way as the test and standard. Any value obtained for the blank is, of course, subtracted

from the value obtained for the blank is, of course, subtracted from the value for the test andstandard in the final calculation, since the blank value is due to the reagents used and not the

substance under investigation. The practical use of blanks and standards is well illustrated

in the numerous colorimetric estimations in this volume. Several blanks or controls need to


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be used when working with enzymes, but these are considered separately in the section on

enzymes.

Chloride (mmol/l)

Estimation Experimental Average

Number value value

1 102 102

2 104 1033 106 104

4 104 104

5 103 1046 105 104

Table 1.4 Determination of serum chloride

Random errors. Finally there are random errors which are individually unpredictable.These are seen when one person carries out a number of determinations under identical

conditions and obtains a slightly different result each time. This random error can be

considerably reduced by taking a large number of measurements and calculating the average

value (Table 1.4). For many purposes, duplicate estimations are sufficient provided there isgood agreement between them, and this is usually the situation for readings obtained in, say,

the construction of a calibration curve. The degree of agreement between replicate

experiments is termed the precision. Precision does not mean accuracy, since measurementsmay be highly precise but inaccurate due to a faulty instrument or technique.

In many cases, however, the precision is not as good as that shown in the example (Table1.4) and there is a much greater spread of results. It is, therefore, useful to be able to give

some measure of the spread of readings obtained and, in order to do this, some elementary

concepts in statistics are now introduced.

A number of equations are given which are obtained from the theory of statistics and these

equations are used as tools with no attempt made to derive them.


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The Normal Distribution Curve

If a very large number of readings are taken of some quantity (x) then a curve can beconstructed which shows the number of readings related to the value of x (Fig. 1.2). This

normal, or

Fig. 1.2 A normal or Gaussian distribution of readings

Gaussian, distribution of results has a number of characteristics:

1. The mean value is x and the curve is symmetrical and has a maximum at this value.

2. The point of inflexion occurs at x + o and x - o so that 68% of all values lie in the

cross-hatched range x o.

3. The curve is such that 95% of all values will be in the shaded range x 2o and 99%

of all values in the range x 3o.

Standard deviation (SD). The value of o is the standard deviation and is a measure of the

spread of expected results. o can be calculated from the individual results (x1, x2, x3 ....., xn),

the number of readings taken (n) and the mean (x).Now

x = (x1 + x2 + x3 + ... + xn)/n = xn/n.

If the deviation of each sample from the mean is represented by d, then:

d1 = x1 - x,d2 = x2 - x,

d3 = x3 - x,

dn = xn - x.

The sum of the squared deviations is known as the deviance, and, where this is divided by

the number of samples, then the variance (o2) is obtained.

o2

= (d2

+ d2

+ ... + d2)/n

The square root of the variance than gives the standard deviation (o).

Now the mean x and the standard deviation (o) cannot be known precisely, unless an infinite

number of measurements are made. In practice, only a limited number of measurements arepossible and a close estimate of the variance of the variance of the population can be

calculated from a finite number of readings by dividing the deviance by the number of

degrees of freedom (n - 1), rather than by the size of the sample (n).

It is tedious to calculate the deviation of each individual reading from the mean and the

variance, and hence the standard deviation can be more readily determined from the sum of

the values of x (x) and the sum of the values of x2

(x2) using the following working

formulae:


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o2

= x2

- (x)2/n

n - 1

Standard error of the mean (SEM). Usually, however, a mean value is obtained from theindividual readings and it is more important to know how far this value lies from the

unknown mean of the whole population than to know the spread of results. The standarderror of the mean (om) gives an estimate of the probable error in determining the mean of thepopulation from a finite number of samples.

om = o/ n.

From this it can be seen that the larger the number of samples then the smaller will be the

SEM and the closer to the 'true' mean of an infinite number of readings.

Biological variation

A physical quantity, such as the density or viscosity of a pure liquid, can be measured in thelaboratory and the value obtained compared with the correct figure. Some random variationwill be observed, but with care this should be very small, so that only a few measurements

need to be taken and the mean calculated. However, this is not the case for many

measurements made in biology, where there is often no single 'true' value but a range of so-called 'normal' values. For many measurements a symmetrical 'normal' type of distribution

is followed and simple statistical methods can be applied. In this case, the normal range is

usually taken to start at (x - 2o) and extend to (x + 2o), which would include 95% of allvalues (Fig. 1.2). Sometimes a 'skewed' distribution is seen which requires more complex

mathematical treatment.

The Student t test. In some biochemical experiments it is important to know whether an

experimental has caused a significant change in a measured quantity or whether the value

obtained is due to chance. An English statistician, who signed himself 'Student', devised a

simple test for determining the probability whether a sample belongs to a given populationor not by taking into account the spread of results often found.

We have seen that, for a very large number of measurements, the standard error of the meanis close to that of the population (x om), but most experiments involve relatively small

samples and the mean of the sample (m) is related to the true mean:

m = x t. om

Or

m = x t. o/ n,

where s is the standard deviation of the sample and n the number of measurements taken.

Statistical tables have been prepared, and the probability of t can be looked up using the

number of degrees of freedom (n - 1). A probability of 0.05 means that there is a 5% chancethat the sample is the same as the population. Similarly if the value of t falls under the 0.01

level of probability then this means that there is only a 1% chance that the sample is the

same as the population. These two levels are the ones usually adopted as the confidence


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limits in biology. The results are considered to be significant at the 0.05 level and highly

significant at the 0.01 level of probability.

Two samples consisting of a finite number of measurements each can be compared directly

and a value of t obtained using the following equation:

t = (x1 -x2)/{(n1 - 1)s2

+ (n2 - 1)s2

1 1)}1/2

n1 + n2 - 2 n2 n2)}

Volumetric glassware

Cleaning of glassware. Scrupulously clean glassware is essential for accurate work. After

use, apparatus is washed in warm water containing soap or detergent then rinsed thoroughly

in tap and distilled water. Excess of detergent should be avoided since this may interferewith some of the experiments. Dirty apparatus is cleaned by first removing grease with a

rag soaked in chloroform or benzene, then by soaking overnight in chromic acid. Very dirty

apparatus can be cleaned by soaking in a mixture of concentrated nitric and sulphuric acid ifchromic acid is not effective. All traces of the cleaner are then removed by repeated rinsing

in tap water followed by several rinses in distilled water. Normal glassware is then dried in

an oven, but volumetric glassware should not be heated but rinsed with small volumes of

alcohol, then ether, and finally dried in a stream of warm air.

Types of pipettes. Pipettes are designed class A or B according to their accuracy. Class A

pipettes are most accurate and the tolerance limits are well defined, i.e., 0.01, 0.02,and 0.04 ml for 2, 25 and 50 ml pipettes respectively. A class A pipette with a certificate

of calibration issued by the National Physical Laboratory is the most accurate of all. Class B

pipettes are less accurate but quite satisfactory for most purposes, especially when calibrated

by the user. Calibration is readily carried out by filling the pipette with distilled water atroom temperature, then allowing the contents to drain into a weighed bottle. Stopper the

bottle and reweigh and carefully note the temperature of the water. The capacity of the

pipette can then be calculated from knowledge of the volumes of known weights of water atvarious temperatures (see Vogel, A. I. Textbook of Quantitative Inorganic Analysis).

Obviously, significant errors will result if the temperature of the liquid pipetted is widely

different from the temperature of


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18

Fig. 1.3 Normal analytical pipettes

calibration. The usual temperature of calibration is 20C and this is marked on the pipette.

Pipettes to deliver. These are designated D for deliver and are bulb pipettes (Fig. 1.3(a)).The pipette is first rinsed several times with a little of the solution to be used, then filled tojust above the mark. The liquid is allowed to fall to the mark and the tip is carefully wiped

with filter paper. The contents are allowed to drain into the appropriate vessel. After the

flow of liquid has ceased, the jet is held against the wall for 15 s, then removed.

With accurate pipettes, the drainage time is defined and marked on the bulb. A certain

amount of liquid will remain at the tip and this must not be blown out.

Graduated pipettes. These consist of a glass tube of uniform bore with marks evenlyspaced along the length. The interval between the calibration marks depends upon the size

of the pipette (Fig. 1.3(b)). They are commonly used in biochemistry for measuring out oddquantities. The liquid is delivered by allowing it to fall from one calibration mark toanother. Volumes can be delivered with reasonable accuracy providing due care is taken. It

is better to use a 1 ml graduated pipette to deliver 0.9 ml rather than a 10 ml pipette. Most

of these pipettes are calibrated so that some liquid is left at the tip and, like the bulb pipette,this is not blown out. Some serological pipettes have a ground glass band at the top and, in

this case, the last drop of liquid has to be blown out for accurate measurement. Another

modification of this type of pipette has only two marks accurately established, often eitherside of a small bulb (Fig. 1.4(a)).


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Fig. 1.4 Micro-pipettes

Micro-pipettes. These are frequently used in biochemical estimations, since often onlysmall quantities of material are available for measurement. Blood or serum is oftenmeasured and, when such viscous fluids are used, an Ostwald - Folin type of pipette is

convenient. These pipettes have less surface per unit volume than the usual pipette, no

sharp shoulders to hinder drainage and capillary tubing for the stem. In an ordinary pipette,blood is left on the wall and in the jet so that less is delivered than should be. For quantities

of the order of 0.1 ml, this error is appreciable. This problem can be overcome to a certain

extent by using an Ostwald-Folin blow out pipette (Fig. 1.4(b). These are marked 'B' for

blow out, which should not be confused with class B pipettes calibrated to contain, and aredesignated by a ground glass band at the top.

Blood and other viscous fluids are more usually measured with pipettes calibrated tocontain. These are marked (In or C) for contain and their appearance resembles the

Ostwald-Folin blow out (Fig. 1.4(c)). A pipette with a straight bore (Fig. 1.4(d)) is better,

since small bubbles of air may be trapped in the bulb when blood enters the stem. The

pipette is calibrated at 0.1 mlor0.2 ml and is often backed with white enamel. Pipettes withan automatic zero are now widely used (Fig. 1.4(e)). A constriction incorporated into the

bore, so that once filled the fluid will drain to the constriction but not beyond it and this type

is now ideal for measuring out microlitre quantities accurately. All three pipettes are used ina similar manner. Filling is carried out as usual and, after carefully wiping the outside, the

contents are rinsed several times into a known quantity of reagent and finally blown out.

A final work of warning; some Ostwald-Folin pipettes are calibrated to deliver and aremarked EX or D. They should not be confused with those previously described (Fig. 1.4(b),

(c)).

Microsyringes are also commonly used. In biochemical work for delivering small quantities

of liquid.

Burettes. These deliver odd quantities of liquid accurately and, as such, are used in

volumetric titrations. Microburettes of 1, 2, and 5 ml capacities are commonly used in

biochemical analysis and may be calibrated in a similar manner to pipettes. Burettes of

these capacities should have narrow jets so that each drop of liquid delivered is as small avolume as practicable. An error of two drops (0.1 ml) in a volumetric titration involving 20

ml is not very great, but in a titration of about 1 ml it is quite considerable. Microburettes

have a narrow bore and the liquid should be given time to drain to the required mark before

taking the reading. As with pipettes, any liquid remaining at the tip should be removed bytouching it lightly against the receiving vessel.

Measuring cylinders. The measuring cylinder is often misused in teaching laboratories and

has earned the name of 'the lazy student's pipette'. It is not a substitute for the pipette or

burette, since it does not deliver the stated volume, but only measures. It can, however, beused to deliver relatively large volumes when accuracy is not important.


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20

Volumetric flasks. Volumetric flasks are calibrated to contain the volume specified at a

fixed temperature, usually 20C. A good flask will have a narrow neck and a thin etchedline extending completely round the neck. This enables an accurate adjustment of the liquidlevel to be made and avoids errors due to parallax. Calibration is effected by weighing the

flask empty, filling with distilled water at room temperature and reweighing. The volume

contained in the flask is then calculated as previously described. Drops of liquid adhering to

the glass above the calibration mark should be removed since this leads to errors.

Before making up to the mark with solvent, it is important to see that all solid is dissolved

first. If difficulty is encountered in dissolving a compound, then the suspension should first

be heated in a breaker and cooled to room temperature before being transferred to thevolumetric flask. Once a volumetric flask is heated it becomes simply a flask and is no

longer volumetric, so it must not be dried in the oven.


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21

Chromatographic Techniques

Plant pigments like chlorophyll, carotenes and xanthophylls can be separated by

techniques such as paper chromatography and column chromatography. In this

laboratory you will use these techniques to separate compounds that were extractedfrom the leaves of the croton plant ( Croton glandulosus )

Reagents and equipment needed:-

a) 95% alcohol, petroleum ether, hexane, aluminum oxide, filter paper, hot plates and

20-25 small chromatographic columns.

A. Boil 10g croton leaves (chop the leaves into small pieces) in 20 ml of 95%

alcohol in a suitable beaker. This MUSTbe done using a Hot Plate and NOT a

Bunsen Burner. Boil until the solution is dark green.

B. Place 5 ml of the solution in a suitable test tube and add 5 ml of hexane. Corkthe tube and shake well. Let stand in a test tube rack ( for at least 1520 min)

and observe for separation into two(2) layers. Addition of water, drop wise,with shaking will aid in the separation process.

Questions

1. What compounds do you think the upper layer contain?

2. What compounds do you think the lower layer contain?

3. Which of these compounds is essential for higher plant photosynthesis?

C. Paper ChromatographyEach group of students, will be given a strip of filter paper, which is long enough

to protrude one (1) inch above the rim of these test tube. Cut a point at one end

of the filter paper. Using a capillary tube carefully apply one drop of the

alcohol extract obtained from Section A to a point one (1) inch from thepointed end of the filter paper.

Be sure the spot is centered on the paper and allow it to air dry.

Add 2 ml of a solvent mixture (10% acetone: 90% petroleum ether) to the test

tube. With the spot dry, insert the paper strip into the test tube with the pointedend down. Be sure that the spot is above the solvent (Why?) and do not let the

lower edges of the paper strip touch the test tube wall. Cork the test tube

and allow the solvent front to move within one (1) inch from the level of the

cork. Carefully remove the paper strip and allow it to air dry. Outline theposition of the pigments. Try and identify (with help from the demonstrators)

the spots.

Draw a line at the solvent front.Measure the Rf.


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Questions

1. Chromatography is an extensively used method for the separation and

identification of mixtures of substances in solution. What are the principlesupon which this method is based?

D. Column chromatography

Each group will be provided with 1) a small chromatographic column; 2) about

1015g aluminum oxide (for chromatographic use) to pack the column; a

small piece of glass wool.

Insert a small piece of glass wool to the end of the column containing a rubber

bung. Make slurry by mixing the aluminum oxide with 95% alcohol. With the

column held in a vertical position using a column stand or clamp, pack thecolumn with the slurry. The pinch clamp that is fixed to the rubber tubing can

adjust the flow rate through the column. Allow the column to pack properly; let

the column packing occupy of the column length.

Apply 1 ml of the alcohol extract from section A into the column and run the

solution into the bed of the column. To a test tube rack add five (5) serially

numbered test tubes. Elute the column with 95% alcohol collecting five (5) 4mlfractions.; this will be scanned (by the demonstrator) using a scanning

spectrophotometer between 380-700nm. The result will be given to you for

interpretation

.

Questions

1. Did the column separate the components of the solution? Are the basic

principles of paper chromatography different from that of columnchromatography ?

2. If there is a bluish band at the top of the column, signifying the presence of

another component(s), how could you remove it from the column?

3. Based on the results of the scan and your knowledge of the plant pigmentsbeing examined, can you now positively identify the compound?

4. Calculate the retention factor for each spot on the chromatogram

5. What additional steps could be taken in the experiment to identify the spots?

6. What are the principles of chromatography?


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23

ProteinI

Objectives

To become familiar with three common methods for the estimation of the protein content of

an aqueous solution.

Introduction

The estimation of the amount of protein contained in an aqueous solution is one of the mostcommon procedures performed in scientific research. Indeed, the original paper describingthe Lowry procedure is one of, if not the most cited research articles in the whole of

scientific history!

The three methods to be used in this experiment (the Biuret, Lowry and Bradford methods)

are applicable to the estimation of relatively low levels of protein in aqueous solutions, such

as are commonly encountered in protein isolation or characterization experiments. A

different method is normally used when the estimation of protein nutrient content isrequired, such as in foodstuffs, and this is the Kjeldahl method of total nitrogen estimation,

which involves the digestion of the entire sample with excess conc. sulfuric acid and

determination of the ammonia so produced.

There is also a method applicable in the same circumstances as would call for the three of

choice here: a method which relies on the estimation of the aromatic amino acid residues

normally contained in proteins by UV spectroscopy at 280 nm (with a correction at 260 nmto account for the possible presence of nucleic acid in the sample). This method (that of

Warburg and Christian) is beyond possibility at the moment in this class.

Both the Biuret and Lowry methods depend upon the formation of a blue-coloured complex

when copper in alkaline solution reacts with proteins, whereas in the Bradford method max

of a dye shifts when it binds to protein.

Reagents

Bovine serum albumin (BSA) @ 10 mg/ml in water (For Standard solutions)

Biuret

1.50 gm cupric sulfate (CuSO4.5H2O)

6.00 gm sodium potassium tartrate (Na+K

+C4H4O6.4H2O)

dissolved in 500 ml water in 1000 ml volumetric flaskwith constant swirling add: 300 ml of 10% NaOH (prepared from

stock, carbonate-free 65-75% NaOH).


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Make up to mark with water. Discard if any precipitate forms.

Lowry

Mix together in a beaker:

1.5 ml of 2% (w/v) sodium potassium tartrate and1.5 ml of 1% (w/v) cupuric sulfate and

150 ml of 2% (w/v) Na2CO3 solution in 0.1 N NaOH

Phenol reagent (purchased ready mixed)

Bradford

100 mg Coomassie (or Kumasi depending upon how the Ghanian city is spelled by thesupplier) Brilliant Blue G-250 dissolved in 50 ml 95% ethanol. To this add 100 ml of 85%

(w/v) phosphoric acid and dilute to 1 litre.

METHODS

Under normal circumstances these methods of estimation require that a calibration curve beprepared by dilution of a standard solution of protein and the protein content of the samples

are determined by comparison to this curve. The conventional protein standard to use is

BSA which is cheap and shows relatively little variation from one supplier's batch toanother. Here only the calibration curves will be prepared, however an important principle

must be outlined. The colour response of most if not all colourimetric estimations varies.

This variation is due to many different factors, a few of which are given here: differences in

the preparation of stock reagents, and their mixing: differences in the execution ofprocedures, age of reagents, instrument variation etc. Thus unless a particular colorimetric

estimation is performed with a great frequency it is necessary to perform a calibration each

time that the reagents are used. If estimation becomes routine then the necessity to calibratethe system is reduced by the experience of the operator.


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I T IS CRITICAL THAT ALL MI XING OF SAMPLES ANDREAGENT BE DONE THOROUGHLY

Biuret estimation:

Using the 10 mg/ml BSA and the necessary dilutions with water, prepare a series of

duplicate standards with the following protein concentrations in 1 ml:

0.0; 0.1; 0.5; 1.0; 2.0; 4.0; 6.0; 8.0; 10.0 mg/ml

1.0 ml standard/samplemixed with 4.0 ml reagent, leave at room temperature for 30 min

and read OD at 540 nm.

Lowry estimation:

Using the BSA prepare a series of duplicate standards with the following concentrations in

1.0 ml:

0.00; 0.05; 0.10; 0.15; 0.20; 0.40; 0.60; 0.80; 1.00 mg/ml

1.0 ml standard/sample mixed with 4.0 ml of copper alkali solution, leave for 10 min atroom temperature.

Mix with 0.5 ml phenol reagent and leave for at least 30 min. Read OD at 740 nm.

Bradford estimation:

Using the BSA prepare a series of duplicate standards with the following concentrations in

0.1 ml:

0.00; 0.05; 0.10; 0.15; 0.20; 0.40; 0.60; 0.80; 1.00 mg/ml

0.1 ml standard/sample mixed with 5 ml of reagent and read OD at 595 nm after 2 min but

before 60 min.

RESULTS

1. Plot the three calibration curves that you have obtained on separate sheets of graph

paper.

2. Compare and discuss the three methods under the headings of

(a) ease of execution (b) sensitivity


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26

Yeast Invertase

Introduction: Yeast invertase catalyses the following reaction:

invertaseSucrose glucose + fructose

Activity of the enzyme can be determined by monitoring the formation of glucose.

Commercially available kits (eg. Worthington statzyme glucose, "WSG") can be used forthis purpose. They contain 2 enzymes and one or more chromogens which together promote

2 successive reactions, viz:

glucose oxidaseD-glucose + O2 + H2O2 H2O2 + D-gluconic acid

H2O2 + chromogen + p-hydroxybenzoate coloured complex + H2O

The absorbance of the coloured complex solution (at 500 nm) is directly proportional to the

concentration of glucose, and is measured using a spectrophotometer. Conversion of

absorbance to concentration can be carried out using a calibration graph. The concentrationsof glucose are determined. The former (y-axis) are plotted against the latter (x-axis).

The objectives of this experiment are (i) to extract and partially purify invertase; (ii) to study

the effect of enzyme concentration, pH, temperature, substrate concentration and inhibitorson invertase activity.

The experiment will take 2 days to complete. It is not important in which order the varioussections are done. You should, however, try to complete at least 3 sections on the first day.

Method

A. Preparation of invertase. Take about 1 g dried yeast, add about twice this

weight of clean sand and 20 mL water. Grind the mixture thoroughly in a mortar.Decant 5 mL of liquid into a 50 mL centrifuge tube, chill in an ice bath, slowly add

25 mL of 95% ethanol and let stand for 15 min. Centrifuge the suspension and pour

off the supernatant. The enzyme is in the pellet; suspend this in 20 mL of 2mMEDTA solution. Keep in an ice bath as stock before use for each experiment.

B. Calibration Curve. Set up the following in 7 test tubes.


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Tube #1 1 & 2 3 & 4 5 & 6 Blank

_______________________________________________________________________

1.0 mM glucose 0.1 0.3 0.6 0.0 ml

(mL)H2O 0.9 0.7 0.4 1.0 ml

_______________________________________________________________________

Add 4 ml WSG to each, mix and incubate at 37C for 15 min. Measure absorbance of all

samples against H2O at 500 nm. Plot a graph of absorbance (y-axis) vs amount of glucose

( moles).

C. Activity of invertase preparation over time. Set up 5 tubes each containing 8.0ml

H2SO4. Add the following to a conical flask:

0.3 M sucrose 7.5 mL

acetate buffer pH 4.7 4.5 mL

H2O 1.5 mL

Bring to 37C in a water bath; add 1.5ml enzyme preparation and mix. Rapidly transfer2.0ml incubation mixture to one of the test tubes containing H2SO4. Mix well by inverting

tube. Repeat sampling after 3, 6, 12 and 18 min. Remove 0.1ml from each of the 5 testtubes and make up to 1.0ml by adding 0.9ml water. Assay these for glucose by adding

WSG as above.

Plot moles glucose formed in a 2ml incubation mixture vs incubation time.

Note: The 0.1ml aliquot represents a fraction (0.1/10 or 1/100) of the 2ml incubation

mixture which was diluted to 10ml with H2SO4.

D. Effect of [E] on reaction rate. Set up the following in test tubes:

_________________________________________________________________________

Tube No. (1) (2) (3) (4) (5)__(mL)___________________________________________________________________

____

0.3 N sucrose 1.0 1.0 1.0 1.0 1.0

acetate buffer(pH 4.7) 0.6 0.6 0.6 0.6 0.6

water 0.4 0.3 0.2 0.1 -

Equilibrate at 37 (3 min) then add at 1 min intervals

Enzyme 0.0 0.1 0.2 0.3 0.4ml


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Mix and start timing after enzyme addition; exactly 1 min later in each case, add from a

burette 8.0ml of 0.1 M H2SO4.

Take 0.1ml from each, add 0.9ml water and determine glucose. Plot the resulting data as

velocity (v) vs ml of enzyme; (v) must be expressed at moles glucose formed/min or othersuitable time unit.

E. Effect of pH. Into each of 4 test tubes, pipette 1.0ml of 0.3 M sucrose. Add 0.8ml

of acetate buffer pH 3.5 to tube 1 and 0.8ml phosphate buffer, pH 6, 7 and 8respectively to tubes 2, 3 and 4. At timed intervals, add 0.2ml enzyme and incubate

for 6 min at 37oC. Stop reaction with 8ml H2SO4 and measure glucose as above.

Combine the data with previous results for pH 4.7 on a graph.

F. Effect of temperature. Design and perform an experiment to show the effect of

temperature on reaction rate. Use at least 4 points in the range 0.80o. Plot your

results on a graph.

G. Effect of [S]. Prepare a range of sucrose concentrations as follows:

Sucrose final conc (M) 0.3 0.1 0.05 0.03 0.02

Vol 0.3 M sucrose (ml) 2.0 2.0 1.0 1.0 0.5

Vol water (ml) 0.0 4.0 5.0 9.0 7.0

Add 1ml of each of the 5 sucrose solutions to a test tube containing 0.8ml acetate

buffer pH 4.7. Preincubate at 370C for 3 min, add 0.2ml enzyme and stop reaction

after 6 min by adding 8ml H2SO4. Assay 0.1ml aliquots for glucose as before.

Prepare 2 graphs of the results (i) velocity (v) vs [S] and (ii) 1/v vs 1/[S].Deduce Vmax and Km from the plots.

H. Effect of inhibitors(A) Potassium Fluoride. Repeat G, replacing 0.2 ml of the 0.8 ml acetate buffer

with 0.2ml 5 mM KF. Incubate for 12 min instead of 6.

(B) Fructose. Repeat the previous experiment but substitute 0.3 M fructose for

KF. Plot results by the double reciprocal method. In all cases, the rate (v) isto be calculated as moles glucose formed per unit of time, and [S] is the

molar concentration of sucrose in the 2.0ml of incubation mixture, eg. tube(5) contains 1/2 x 0.02 M, i.e. 0.01 M sucrose.


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29

Lysozyme Purification by Ion-Exchange

Introduction: Ion exchange is the exchange of ions of like sign between a solution and an

insoluble solid body in contact with it. There are thus two types of ion

exchangers:

(i) Cation exchangers, in which the solid body carries negative charges and positivecharged

soluble cations bind to it.

(ii) Anion exchangers in which the reverse is the case.

Within each of the above categories, the ion exchangers differ according to the acidity, or

basicity, of the immobile charge. The following shows the ion exchangers most often usedin protein purification. (Note: other types of ion exchange resins such as "Dowex" and

"Amberlite" are used in the extraction of micromolecules.

Nature Functional Group

Anion Exchangers

Diethylaminoethyl (DEAE) weak base -OCH2CH2N+H (C2 H5)2

Quaternary amino-ethyl (QAE) strong base -OCH2CH2 N+(C2 H5)2

CH2 CH (OH) CH3

Cation Exchangers

Carboxymethyl (CM) weak acid -OCH2 COO-

Sulphonyl (SP) strong acid -CH2 CH2 CH2 SO3-

_________________________________________________________________________

In this experiment you will be using the cation exchanger CM-cellulose to purify lysozyme

from egg white.

Lysozyme has a pI of 11.0. This is higher than most other proteins. It is also stable at pH

10. This means that of the pH of egg white is raised to 9.5, of all the proteins present,

lysozyme alone will still carry a positive charge. (A minor constituent of egg-white,avidine, behaves similarly).

If one were to use an onion exchanger at pH 10, the lysozyme would not bind whereas all

other proteins would bind. In order to ensure that all of the anionic proteins would bound, alarge quantity of anion exchanger would be required. It is therefore more economical (and,

all other things being equal, therefore better) to use a cation exchanger which will bind only

the lysozyme and leave the other 90% of the egg-white constituents in solution.


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Purification Procedure

Filter an egg white through muslin and dilute five fold with 50 mM Na 2CO3/NaOH buffer

pH 9.5. Store a sample of this on ice for analyses later.

To 20 ml of the diluted egg white add 1.5 g of equilibrated CM-cellulose equilibrated in 50mM Na2CO3/NaOH buffer pH 9.5 and stir gently for 10 mins or place in stoppered tube and

shake. Centrifuge to pellet the cellulose (2000 rpm for 2 mins) and decant the supernatant.

Keep this first supernatant (label it!) on ice for later, resuspend the pellet in 20 ml buffer andstir or shake gently for 5 mins. Centrifuge again and store this second supernatant (label it!)

on ice. Resuspend the pellet with 20ml buffer at pH 10.50 and shake or stir for 10 min.

Centrifuge and decant the supernatant (label it 10.5) which contains the lysozyme. Wash

the pellet of CM cellulose into the container provided so that it may be recycled.

You now have a sample of the original diluted egg white, the first two supernatants, and the

final supernatant.

Assay of Lysozyme Activity

Lysozyme dissolves certain bacteria by cleaving the polysaccharide component of their cellwalls. A suspension of susceptible bacterial cells in the light path of a spectrophotometer

will scatter the incident light away from the detector and thus reduce the light transmittance.

As lysozyme dissolves the bacteria the light scattering effect is reduced and thetransmittance of light increases.

Substrate: Micrococcus lysodiekticus 20 mg/100; 0.1 M phosphate buffer pH 6.2

Place 4.9 ml substrate in a cuvette in a spectrophotometer zeroed with air reading at 540 nm.

The absorbance should read about 0.4 units. Add 0.1 ml of your sample and start timing.

Quickly mix the cuvette thoroughly (by inverting several times) and place in light path.Take OD readings at 10 second intervals for two minutes. If the OD drops too fast to

measure, dilute a portion of your sample by a known amount and try again.

Suggested Dilutions

Egg white solution: 5-fold, 25-fold, 50-foldFirst supernatant: no dilution

Second supernatant: no dilution

pH 10.5 supernatant: no dilution, 10-fold

Plot the results on a graph of OD vs time.

Determine the initial (steady) rate.


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Results

Take as units of activity the drop in OD 540 nm per minute. You will have a value for eachsample. Multiplying by the appropriate factors, determine the total activity present in all

fractions. How much of the activity that you started with did you recover?

Assuming that you recovered less than 100%, where is the remaining activity, and howmight this be recovered?

Assuming that you apparently recovered more activity than you originally started with, howcould one explain this?

Answer the following questions?

(1) What is the function of lysozyme in egg-white?

(2) Describe the mechanism of action of lysozyme.

(3) What other method could be used to purify lysozyme from egg-white using its unusualpI?


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Banana Polyphenoloxidase (Measuring Browning)

One enzyme with two inseparable types of activity is variously termed tyrosinase, mono-

phenoloxidase, polyphenoloxidase, o-diphenol: oxygen oxidoreductase,dihydroxyphenylalanine: oxygen oxidoreductase.

This enzyme (EC 1.10.3.1) is responsible for the enzymatic browning, which occurs inmany fresh fruits and vegetables when they are damaged. This is due to the aerobic

oxidation of various ortho-diphenols to the corresponding quinones; these highly reactive

products react non-enzymatically to form melanin pigments. The melanins that occur in

foods are formed by polymerization of theo-quinones acid/or by condensation of the quinones with amino acids, peptides on proteins.

The reactions below show the likely sequence of events when dopamine - present in bananas

- is the primary substrate.

In one type of assay, the rate of production of the red compound 2,3-dihydroindole 5, 6-

quinone from dopamine is measured by spectrophotometry (Absorption max, 470 nm) Em =

2512.

An alternative assay measures oxygen consumption, the rate of which is determined by the

oxygen electrode is also a good index of enzyme activity (100% air saturated water contains0.23 moles 02/ml).

Reagents:

Buffers: a) 0.02 M Na phosphate buffer containing1% Triton x - 100, pH 8.0

b) 0.1 M Na phosphate buffer, pH 7.0

Substrates: a) 0.05 M Dopamine HClb) Catechol 0.05 M

c) 3, 4-dihydroxy phenylalanine

(DOPA) 0.05 M

Inhibitors: a) EDTA 30 mM

b) 1, 10-Phenanthroline hydrochloride 30 mMc) Diethyldithiocarbamic acid 30 mM

Other reagents: Ascorbic acid 1.0 mM


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Experimental Procedure

The enzyme is readily extracted from banana pulp with a buffer containing 1% of thedetergent Triton x-100 to protect the enzyme from precipitation by tannins. Buffer is 0.02

M Na phosphate pH 8.0.

Weigh out 4 gm of banana pulp, to which 9 volumes of buffer with 1% Triton x-100 areadded. Homogenize thoroughly by grinding in a mortar. Centrifuge at 20,000 x g for 15

min. Save the supernatant: keep on ice.

+ Enzyme assay: Spectrophotometric method. Try different levels of enzyme extract as

follows:-

0.1 M Na Phos, pH 7.0 1.5 ml

Enzyme 0.2 ml* H20 1.0 ml

0.05 M Dopamine HCL 0.3 ml

Add buffer (0.1 M Na phos, pH 7.0), enzyme and water and observe any changes in

absorbance. Start the reaction by adding the substrate (0.05 M Dopamine HCL) and note....A470 over a period of 3-5 min. Select a period when the.... A470 is linear with time, as the

initial reaction rate.

(1) Use two additional substrates (catechol and 3,4 dihydrophenylalamine) with a fixedamount of enzyme extract, and compare enzyme activities for the various substrates.

(2) Effect of inhibitors. *Replace 1.0 ml of water in incubation mixture with 1.0 ml ofthree inhibitors, at two quite different levels, e.g. 1.0 mM, 10 mM.

(3) Heat an aliquot (approx. 2 ml) of the enzyme extract and assay for enzyme activityusing Dopamine HCl as the substrate.

Boil enzyme extract in a water bath at 100C for 5 min.

+ If enzyme extract has too much enzyme activity, dilute 1 in 5


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34

Polyacrylamide Gel Electrophoresis (PAGE)

Introduction: Most biological polymers are electrically charged and will therefore move

in an electric field. The movement of particles through a solvent by an electric field iscalled electrophoresis. A useful way to characterise macromolecules is by their rate of

movement in an electric field. This property can be used to determine protein MWs, to

distinguish molecules by virtue of their net charge or their shape, to detect amino acidchanges from charged to uncharged residues or vice versa, and to separate different types

of electrophoresis such as zone electrophoresis or continuous electrophoresis. In zone

electrophoresis, a solution is applied as a spot or band and particles migrate through a

solvent that is almost always supported by an inert and homogenous medium such aspaper or in a gel. In continuous electrophoresis, the sample is also applied as a zone

except that it is continuously applied.

The use of gels such as starch, polyacrylamide, agarose and agarose acrylamide assupporting media provides enhanced resolution, particularly for proteins and nucleic

acids. Polyacrylamide gels have replaced starch gels because the amount of molecularsieving can be controlled by the concentration of the gels and the adsorption of proteins is

negligible. Polyacrylamide is currently the most effective support medium in separation

of proteins and small RNA molecules (for nucleic acids that are too large for the

polyacrylamide pores, agarose and agarose acrylamide are superior). Polyacrylamide gelelectrophoretic system for the analysis and separation of macromolecules.

It is not possible to complete all the operations within one day, so special arrangementshave been made (see Notes).

Solutions

A. Running gel for 10.0% PAGE (30 ml)30% acrylamide 10.0 ml

1% bis-acrylamide 3.9 ml1 M Tris

*(pH 8.7) 11.2 ml

SDS 20% 0.15 ml

Water 4.3 ml

TEMED*

10 l

10% ammonium persulfate 100 l

B. Stacking gel (10 ml)30% acrylamide 1.67 ml

1% bis-acrylamide 1.30 ml

1M Tris (pH 6.8) 1.25 ml


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SDS 20% 0.05 ml

Water 5.7 ml

TEMED 5 lC. Sample buffer

2-mercaptoethanol 5%

SDS 2%Tris (pH 6.8) 0.62 MGlycerol 10%

Bromophenol blue (2% in ethanol) 0.02%

D. Running bufferTris 12.12 g

Glycine 57.5 g

SDS 4.0 gAdd water to final vol. of 4 litres

E. Staining solution: 0.02% coomassie blue in methanol: acetic acid: water(45:10:45)

F. Destaining solution: Methanol: acetic acid: water (15:7.5:77.5)

Method

Set up plates of gel apparatus, use petroleum jelly to seal bottom of plates. Pour runninggel solution into plates using small beaker or 10 ml pipette to approximately 1 cm below

top. While pouring the running gel, avoid air bubbles. Overlay gently with 1-2 mm

water with the help of a Pasteur pipette. Leave for 2 h to polymerise and blot off water

with tissue paper.

Place teeth of comb in between the plates before gently adding the stacking gel. Overlay

with water. Leave for 2-3 h to polymerise. After polymerisation, carefully remove thecomb and the bottom seal. Attach the plates to the main slab gel apparatus. This exercise

should be done carefully. Once the plates are fixed tightly to the apparatus, running

buffer should be gently applied (10 - 20 l, approximately 1 mg protein/ml) into the slots

made by the comb. The gel apparatus is now ready to be connected to the power supply.Run at 150 V (constant voltage for 5 - 6 h).


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Notes

You will appreciate that the whole operation cannot be performed in the time available

and so the following will be done in class.

1. Some gels will have been prepared, so all students will apply the samples to thewells and the power connected.

2. All students will practise assembling the glass plates and mixing and pouring therunning gel.

3. Our staff will continue to run the power until electrophoresis is achieved, removethe gels, stain with coomassie blue for 2 - 3 h and destain them for you overnight.

4. You will inspect as much of the process as possible.5. Your report will be judged on your explanation of the theory of gel

electrophoresis and the practical pitfalls to be avoided.


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Respiratory Control & ATP Synthesis in Mitochondria

Objectives

1. To set up and use an oxygen electrode for measuring oxygen consumption from asolution.

2. To measure oxygen uptake by mitochondria, and demonstrate control of ATP

synthesis by ADP + P1 (respiratory control).

3. To study the effects of different inhibitors on respiration and ATP synthesis with

different substrates.

The Oxygen Electrode

Introduction And Setting Up as before (see earlier expt. on yeast)

The sample chamber should be thermostatted between 30 & 370C in this experiment.

Preparation Of Mitochondria

Mitochondria will be prepared for you from rat liver. The procedure involves rapidlyremoving the liver from a freshly killed rat, homogenizing at 0

oC in a pH 7 buffer, and then

differential centrifugation to remove nucleic and cell debris (low speed) and mitochondria

(higher speed), all at 0oC.

Mitochondria are alive and will die as time goes on. KEEP YOUR SUSPENSION IN ICE

WHEN NOT IN USE, to cut down this disintegration.

Solutions

0.5 M succinate0.5 M glutamate

0.3 M NADH

5 mM ADP

60 mM KCN3 mg/ml rotenone

mg/ml oligomycin6 mM 2, 4 dinitrophenol

mitochondrial reaction medium -

150 mM KCL, 10 mM potassium phosphate, 5 mM Mg C12, 20 mM MOPS(3-[N morpholino) propane sulphonic acid) brought to pH 7.4.


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Experimental

1) Set up oxygen electrode and check it is working as described.

2) Pipette 2 ml reaction medium and 0.1 ml mitochondria into reaction chamber.Follow oxygen uptake rate (% decrease/min) by timing fall of meter between 2points on the scale. This rate (the endogenous rate of respiration) is due to oxidation

of substrates within the mitochondria, and should be low (< 2% per minute).

3) Add 0.1 ml succinate, and again follow the oxidation rate, by taking the meter

readings every 30 secs for 3 min.

4) Add 0.1 ml ADP and follow the meter readings at 15 sec intervals for about 5 min.There should be an initial increase in rate after ADP addition, followed by a decline

to the original rate.

5) If the rates were too high to measure easily use 0.05 rather 0.1 ml mitochondria in

subsequent measurements. If too low, try 0.2 ml mitochondria.

6) Plot the % 02 at the various times of measurement on a graph of % 02 vs time. Thegraph should look like this:

Draw the best straight lines through sections 1, 2, 3 and 4, and calculate the total % 0 2 usedup during period (3).

7) Given that 100% air saturated water contains 0.23 mmol 02/ml, calculate the rate of

oxygen uptake (in mmol 02/min) during phases 1 - 4.

The ratio (3)/(2) i.e. rate + ADP / rate - ADP is called the respiratory control index, and is

high (< 5) in well coupled mitochondria. Calculate this ratio for this and all other substrates

used.

Also, from the % 02 used up during period (3), you can calculate the total number of mols of

0 (not 02) used during ATP synthesis (since, when the rate slows down again, all the ADPhas been converted into ATP). The number of moles ATP made (= no of mols ADP added)

divided by mols of 0 used up is the P/O ratio (see text book). Calculate this ratio for this and

other substrates.

8) Repeat this experiment, using glutamate as substrate in place of succinate. Compare

the P/O ratio, and the respiratory control index with the 2 different substrates, and

comment on your results.


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Actions of Inhibitors and Uncouplers

1) Carry out the above experiment again, but varying the order of substrate addition etc,

as shown below. Make sure your last addition is made before the needle drops

below 15%. Between each run, wash out the chamber well with distilled water (4 -5 times, also wash the stopper well, first under the tap then with distilled water, toremove all traces of the inhibitors.

A. Buffer succinate oligomycin ADP dinitrophenol KCN

+ mitos (0.1 ml) (50 l) (0.1 ml) (50 l) (50 l )

B. Buffer succinate dinitrophenol ADP rotenone KCN

+ mitos (0.1 ml) (50 l) (0.1 ml) (50 l) (50 l)

C. Buffer glutamate dinitrophenol rotenone succinate KCN+ mitos (0.1 ml) (50 l) (50 l) (50 l) (50 l)

D. Buffer succinate KCN ADP dinitrophenol

+ mitos (0.1 ml) (50 l) (0.1 ml) (50 l)

E. Buffer ADP NADH glutamate

+ mitos (0.1 ml) (0.1 ml) (0.1 ml)

Runs A and D compare the effects of oligomycin and KCN, two inhibitors of

phosphorylation. What conclusions can you draw from these experiments as to the sites ofaction of these compounds? Why does dinitrophenol release inhibition due to oligomycin?

Run C compares the effect of 2 electron transfer inhibitors, rotenone and cyanide, on the

oxidation of glutamate and succinate. Why do they affect the different substratesdifferently?

Run B allows comparison of the respiratory control ratio (rate + ADP/rate - ADP) measured

previously with the same ratio measured with dinitrophenol present. Why do the 2 ratiosdiffer? What does dinitrophenol alone do to the oxidation rate, and by what mechanism

does it act?

Run E compares the effect on respiration of adding NADH external to the mitochondria, andgenerating it inside from glutamate. Why are the oxidation rates different in the 2 cases?

Why is ADP added in this experiment? By what reactions inside the mitochondria does

glutamate generate NADH? How does cytoplasmic NADH normally get inside themitochondria?


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40

Affinity Chromatography of Lactate Dehydrogenase

Introduction

The high specificity, rapid reversibility and reasonable strength of the interaction between amacromolecule and its Ligand are fundamental to the efficiency of biochemical systems.(Ligand, literally that which binds, can be substrate, modular, inhibitor, etc.). This

efficiency is harnessed in the technique of affinity chromatography whereby a Ligand is

immobilised by covalent linkage macromolecule is allowed to interact with the immobilisedLigand, usually in a column, and subsequent washing of the column removes all

components of the crude preparation that are not bound to the column. The bound species

can then be recovered by competing with the binding interaction: either specifically, by

washing the column with non-immobilised Ligand or in a non-specific manner by washingthe column with a high ionic strength solution to disrupt the electrostatic interaction between

immobilised Ligand and macromolecule.

If the Ligand is NAD+, for instance, this is called a group-specific Ligand because a group

of macromolecules will bind it (NAD+

-linked oxidoreductases). Thus, if a crude tissue

supernatant were applied to such a column, most if not all of the group of proteins will

remain bound to the column. Specific elution of the desired proteins can theoretically beachieved by eluting the column with an [NAD

+] gradient, each protein being eluted

according to its affinity constant.

In this experiment, a group-specified Ligand, Procion Red or Blue, will be used to purify

lactate dehydrogenase from rat liver supernatant. In the interests of economy, 1 M NaCl

will be used to recover the bound enzyme.

The amount of accessible Ligand that is immobilised to the column will determine the

amount of interacting proteins that can be retained by the column. Overloading will result in

the appearance of potentially binding protein in the washout peak of elution.

Method

Four grams of rat liver were homogenized in 40 ml ice cold 10 mM K+phosphate buffer, pH

7.0 (buffer A) and centrifuged for 30 min at 25,000 rpm. 38 ml of supernatant (S) were

recovered.

Set up the micro-column as follows:

1. Lightly tamp a small plug of glass wool into the pasteur pipette with the fineglass rods provided. If the plug is too hard, you will get a very slow flow rate - startagain.

If the plug is too loose, the gel will elute as well as buffer - start again.2. Attach tubing to the pipette and gently grip the tubing with the clamp.


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3. Clamp the pipette into a burette stand.

4. Fill the column with water and adjust the flow rate with the tubing clamp.

5. Introduce gel slung into the column until the bed is just below the constriction in theglass.

6. Wash the column with at least 3 bed volumes of buffer A (WITHOUT THE 1 M

NaCl !!!)

NEVER LET THE GEL BED RUN DRY. If it does, start again.

Now apply 0.2 mls (S) to the column allowing it to just run into the surface of the bed.Allow it to equilibrate for about 5 minutes.

Place a measuring cylinder under the outlet.

Wash the column with 6.0 mls of buffer A (NO NaCl), collecting three 2.0 ml fractions (F1,

F2, F3).

Wash the column with 6.0 mls of buffer A containing 1 M NaCl collecting three 2.0 ml

fractions (F4, F5, F6).

After appropriate dilutions you will now determine protein content and LDH activity in (S)

and F (1-6).

Lactase Dehydrogenase Assay

Pyruvate condenses with dinitrophenylhydrazine to form a DNP hydrazone, and when this is

made alkaline a colour develops. A standard solution of pyruvate is used to construct acalibration curve, against which to determine the amount of pyruvate formed enzymically

from the lactate.

The calibration curve and enzyme assays should be performed simultaneously AND IN

DUPLICATE !!!

This is the time consuming part of the experiment, prepare as much as possible while thecolumn is running.


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Calibration (all volumes in ml)

________________________

1 2 3 4 5 6________________________

Pyruvate standard 0.0 0.1 0.2 0.3 0.4 0.5

Substrate solution 1.0 0.9 0.8 0.7 0.6 0.5Distilled water 0.3 0.3 0.3 0.3 0.3 0.3DNP hydrazine 1.0 1.0 1.0 1.0 1.0 1.0

________________________

Mix well, incubate at 37C for 20 min, then stop reaction with 0.4M NaOH

10 10 10 10 10 10

________________________

Mix well, leave at room temperature for 10 min.

Read OD at 440 nm.

Enzyme assay (all volumes in ml)

Dilute (S) 1:20, (F1-6) 1:10

________________________________________________Control (S) (F1) (F2) (F3) (F4) (F5) (F6)

(S) 20 10 10 10 10 10 10

________________________________________________

Substrate

solution 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Sample 0.1 (S) 0.1 0.1 0.1 0.1 0.1 0.1 0.1

20

Distilledwater 0.2 - - - - - - -

NAD+

Solution - 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Mix well, incubate at 37C for 15 min, then add

DNP

hydrazine 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Mix well, incubate at 37C for 20 min, then stop reaction with 0.4 M NaOH

10 10 10 10 10 10 10 10

_________________________________________Mix well, incubate at room temperature for 10 min. Read OD at 440 nm.


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Draw a calibration curve of OD vs ml pyruvate solution.

Subtract OD of control from all assays.

Read off the unknown values in mls pyruvate.

Assuming that the relationship between mls pyruvate and moles pyruvate formed/min islinear, and that 0.3 mls pyruvate = 0.5, moles/min, calculate the activity of LDH in each

fraction.


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Results

You should now have enough information to fill in the following table:

_________________________________________________________________________

Volume Protein Activity *Specific Activity Purification Total RecoverySample ml mg/ml mmol/min/ml Factor Activity of

mmol/ min/mg mmol/min Activity_____ ______ _________

(100%)

S

F1

F2

F3

F4

F5

F6

Volume ml - record exact volume of each fraction

Protein mg/ml - remember to take the dilutions into account

Activity mmol/min/ml - remember how much of each fraction was used

in the assay, and by how much each fraction

had been diluted

Specific activitymmol/min/mg - you need to know both the protein concentration and the activity of

each fraction to calculate this

Purification

factor - divide the specific activity of each fraction by that of the supernatant

Total activitymmol/min/ - you need to know both the total volume of each fraction and its

activity to calculate this

Recovery of

activity - since all of the enzyme found in the fractions must have come from

0.5 ml of applied supernatant, what percentage of this Total activitywas found in each fraction?


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Discussion

Find out from the rest of the class whether there was any significant difference between theProcion red and Procion blue. Present the table of results in graphic form using a bar chart,

showing protein concentration, activity and specific activity for each fraction.

You will find that you have apparently recovered more activity than you applied. Think

about it very carefully. Suggest possible explanations and how these might be investigated.

How else might one express the recovery of activity (i.e., as a percentage of what?)

Assessment

You will be marked for overall presentation (i.e. clarity), and for your analysis of the results.

References

1. Affinity Chromatography - Lowe & Dean (1974). In Science Library QP601.L69

2. Methods in Enzymology XXXIV. Affinity techniques Enzyme Purification: Part B

QP601.C733.


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Assay of Enzymes Activity

Quantitative determination of enzyme activity may be made by following the appearance

of products or the disappearance of the reactants in measured periods of time. For many

redox enzymes the uptake of oxygen is measured by means of manometers. In reactions

involving the conversion of oxidized coenzymes to reduce coenzymes, the relativeopacity of the latter, permits an assay technique based on the increase in optical density in

time. NAD+ -approach. Allowing the reaction to proceed in the opposite direction

permits easy assay based on decreasing optical density as a spectrophotometer, aninstrument which measures optical density at specific wave lengths.

Still another assay procedure is afforded by the tendency of certain dyes to change colour

after the uptake or loss of electrons or hydrogens.

A. Such a dye is methylene blue which is coloured (blue) in the oxidized state but

becomes colourless in the reduced state. Thus, the action of the dehydrogenase

might be represented as follows.

(reduced substrate) AH2 M.B H2Oacceptor

M.B. 2H 02

Oxidized substrate A donor acceptor

Reagents: 0.4 M Na Succinate

0.

Documents

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