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Laboratory Exercises in Medical Biochemistry

2nd year, General Medicine

WINTER SEMESTER

2019/2020

Department of Medical Chemistry and Biochemistry

Faculty of Medicine in Pilsen, Charles University

Rules of occupational safety

1. Only practising students, specified by the timetable, are right of entry at the practical classes.

No admittance of any visitors. Authorized personnel only.

2. Students are required to familiarize with their task. Laboratory coats and working instructions are

obligatory. Long hair must be adapted for working with a burner without any risk of injury.

Overgarments and bags must be put on the given place.

3. Any leaving is allowed just with a lecturer´s permission.

4. Only prescribed activities are allowed in laboratories. No eating, no drinking, no smoking and no storing

food in laboratories. Laboratory equipment is not allowed to use for any other purposes.

5. If there is a leakage of harmful chemicals possible, the extraction must be ensured. Working with fuming

substances, substances irritating to the respiratory, toxic gases and vapours, as well as annealing and

combustion is allowed to do just in a fume cupboard.

6. Students must be careful during the manipulation with a safety bulb pipette filler. Pieces of broken glass

must be put in a specific container, label "GLASS".

7. It is possible to pour out only the solvents perfectly miscible with water into the sink. They must be

sufficiently diluted (at least 1:10), maximum of 0.5 litre. Aqueous solutions of acids and alkalis must be

diluted at least 1:30. Solvents immiscible with water, poisons, acids and alkalis over the given

concentration and substances loosing toxic gases and gases irritating to the respiratory must be disposed

into the special waste container.

8. An acid is pouring into the water during the dilution of acids, never vice versa.

9. It is forbidden to suck in solution into a pipette per mouth. A safety bulb pipette filler must be used.

10. Spilt acids must be washed by water immediately, if need be neutralized by sodium carbonate. Spilt

alkalis must be just washed by water.

11. All burners and electrical current must be switched off due to spilling of flammable liquids and it is

necessary to clear the air. Pouring liquids must be absorbed by suitable porous material and it is liquidate

in the appropriate way.

12. During the heating of a liquid in a boiling flask superheating must be prevented by using a boiling chip.

13. It is necessary to check all devices before the start of working. Possible faults and defects must be

reported to a lecturer or a laboratory technician.

14. Intentional handling with electrical device and substances is forbidden. To switch on a device and to light

a burner is allowed by the approval of a lecturer or a laboratory technician.

15. All centrifugation procedures must be controlled by a lecturer or a laboratory technician. Vessels for the

centrifugation must be well balanced and the top of the centrifuge must be closed safely during the

operation.

16. The gas intake and electrical current must be switch off and clear the air if there is a leakage of gaseous

fuels.

17. A lighted burner without supervision is not allowable. If there are any problems with a turner, it is

necessary to switch off the gas intake and the burner must be regulated.

18. Students are obliged to inform a lecturer of any accident, injury, or in case of ingestion chemicals.

19. Serious breach of rules because of a lack of discipline or ignorance is the reason of leaving the practical

classes as an unexcused absence.

20. Students must be informed about classification of toxic, carcinogenic, mutagenic and damaging fertility

substances. Safety sheets of particular substances are available in laboratories.

21. Students must be informed about rules of occupational safety with highly toxic substances (label T+)

using in laboratories (e.g. mercury, potassium cyanide, ethidium bromide, mercury (II) nitrate).

Pilsen, September 16th, 2016 Ing. Václav Babuška, Ph.D.

Deputy Head of the Department

LIST OF EXERCISES

Pipetting training: page 4

Pipetting of distilled water with checking of the precision by weighting

Determination of the density of an unknown solution

Preparation of a solution and its aliquoting into individual tubes

Pipetting of large volumes

Optical methods: page 6

Spectrophotometric estimation of Cu2+ concentration

Identification of acid-base indicator by absorption spectra

Spectrophotometric estimation of Cl- concentration

Chromatography: page 12

Paper chromatography of amino acids

Separation of plant pigments by thin-layer chromatography

Separation of dye mixture by gel chromatography

Potentiometry: page 19

Potentiometric measurement of pH

Demonstration of buffer functioning

Potentiometric titration - estimation of HCl concentration

- estimation of CH3COOH concentration

Determination of the isoelectric point of an amino acid

Osmolality, osmosis, dialysis: page 27

Demonstration of osmosis

Determination of osmolality using cryoscopy

Monitoring the kinetics of dialysis

Antioxidants: page 35

Measurement of total antioxidant capacity (TAC)

Enzymology I: page 40

Enzyme specificity

Dependence of activity of α-amylase on pH

Estimation of alpha-amylase in urine (EPS, Wohlgemuth´s test)

Enzymology II: page 46

Estimation of Michaelis' constant of acid phosphatase

Estimation of catalase activity

Enzymology III: page 50

Estimation of lactate dehydrogenase activity in blood serum

Alanine aminotransferase and aspartate aminotransferase in liver

Estimation of alkaline phosphatase in blood serum

Enzymology IV: page 55

Inhibition of succinate dehydrogenase with malonate

Estimation of aldolase activity

Estimation of xanthine oxidase activity

4

Pipetting training

1) Pipetting of distilled water with checking of the precision by weighting

a) Pipetting of large volumes, pipettes with fixed volume

Place a small empty beaker on a balance pan. The mass of the empty vessel is called the

tare. Press the TARE button to get a reading of 0.000 g.

Take the beaker from the balance, place it on the table and pipette into it distilled water of

the following volumes:

3 x 2000 l

2 x 500 l

For pipetting of 2 ml (=2000 l), use the pipette with the fixed volume. Select the proper tip for the pipette

(big white). To attach the tip, firmly press the shaft of the pipette into the large open end of the tip with light

force to ensure a good seal. Similarly, for pipetting of 0.5 ml (=500 l), use the pipette with the fixed

volume and proper tip.

After you finish the pipetting, place the beaker on a balance pan to read the weight of

distilled water inside. Compare the result with a theoretical expected value obtained as

a sum of pipetted volumes recalculated to mass using the density. For doing this, density

of distilled water at the temparature in the laboratory is =1.000 g/cm3.

Expected volume Expected mass Measured mass

b) Pipetting of small volumes, pipettes with adjustable volume

Place an empty small plastic test tube of a volume 1.5 ml, so called Eppendorf tube, on a

balance pan, and press the TARE button.

Take the open Eppendorf tube in your hand and pipette into it distilled water of the

following volumes:

375 l

25 l

For pipetting of 375 l, use the pipette with adjustable volume in the range 100-1000 l and proper tip.

For pipetting of 25 l, use the pipette with adjustable volume in the range 20-200 l and proper tip (yellow).

After you finish the pipetting, close the Eppendorf tube and place it on a balance pan to

read the weight of distilled water inside. Compare the result with a theoretical expected

value obtained as a sum of pipetted volumes recalculated to mass using the density. For

doing this, density of distilled water at the temparature in the laboratory is =1.000 g/cm3.

Expected volume Expected mass Measured mass

5

2) Determination of the density of an unknown solution

Place an Eppendorf tube on a balance pan and press the TARE button.

Into the Eppendorf tube, pipette exactly 1.000 ml (=1000 l) of a solution, density of

which you want to determine.

For pipetting of 1000 l, use the pipette with adjustable volume in the range 100-1000 l and proper tip.

Close the Eppendorf tube and place it on a balance pan to read the weight of a solution

inside. From a known volume and mass measured, calculate the density.

Volume Mass Density

3) Preparation of a solution and its aliquoting into individual tubes

Pipette into an Eppendorf tube: distilled water 93 l

dye solution 7 l

For pipetting of 93 l, use the pipette with adjustable volume in the range 20-200 l and proper tip (yellow).

For pipetting of 7 l, use the pipette with adjustable volume in the range 0.5-10 l and proper tip (very small

white). At this step, you are adding a very small volume. The best way how to do it: The orifice of the tip

must be dipped below the level of the solution which is already in the Eppendorf tube. By adding these 7 l,

you will simultaneously mix the solution, read further how to do this!

Mix thoroughly the content of the Eppendorf tube. It can be done by so called "pipetting

up and down" several times, i.e. repeatedly pressing and releasing the button of the pipette

causing movement of the solution in the tip "up and down". In this exercise, the solution is

coloured, so you can see what is happening and check if mixed sufficiently. Taking the tip

out of the Eppendorf tube, be careful to make the tip empty!

Prepare 5 microtubes (0.2 ml) in a rack and pipette into each exactly 20 l of the solution

you have prepared.

5 x 20 l

100 l

Evaluate the precision and accuracy of your pipetting!

4) Pipetting of large volumes

Transfer into titration flask: distilled water 10.0 ml

0,1 M HCl 5.0 ml

indicator (methyl red) 5 drops

For pipetting of 10.0 ml distilled water, use the glass pipette and rubber suction bulb. 5 ml of 0.1 M HCl add

from dispenser. "up and down". Adding of 5 drops of indicator as the last step.

6

Optical methods

1) Spectrophotometric estimation of Cu2+ concentration

The hydrated copper complex ion [Cu(H2O)4]2+ is light blue in colour. However, for photometric

estimation, this coloration is of low intensity and it must be highlighted with a suitable reagent like solution of

ammonia that results in formation of dark blue tetraammine copper(II) complex.

[Cu(H2O)4]2+ + 4 NH3 [Cu(NH3)4]2+ + 4 H2O

For determination of the Cu2+ concentration, we will use a calibration curve method. By diluting of

a stock solution (a standard solution of known high concentration) you will get a set of calibration solutions.

Measure absorbances of them at optimal wavelength (absorption spectrum maximum). The calibration curve is

a graph of absorbance as a function of concentration, A = f(c). According to the Beer-Lambert law, it should be

a straight line passing through the origin, i.e. the absorbance (A) is directly proportional to the concentration (c).

Once having a calibration curve, concentration of unknown samples can be easily read from it.

Procedure:

A) Prepare the solutions

Into a test tube rack, place clean and dry test tubes and mark them with numbers:

1 to 10 calibration solutions

0 blank solution

S1, S2, S3, S4 unknown samples

According to the table, prepare the calibration solutions by diluting of the stock solution

of Cu2+ (c = 25 mmol/l) with distilled water. Be careful with pipetting accuracy!

Number Stock solution H2O c(Cu2+)

- ml ml mmol/l

0 - 5.0

1 0.5 4.5

2 1.0 4.0

3 1.5 3.5

4 2.0 3.0

5 2.5 2.5

6 3.0 2.0

7 3.5 1.5

8 4.0 1.0

9 4.5 0.5

10 5.0 -

Transfer by pipetting 5.0 ml of each unknown sample solution into the test tubes marked

S1-S4.

Add 5 ml of ammonia solution into each test tube (blank, calibration solutions, unknown

samples). For doing this, use the automatic dispenser.

Mix the content of all test tubes by turning upside down and back. For closing the tubes,

use the rubber stopper not your thumb!

7

B) Make a search for the optimal wavelength

On a spectrophotometer, measure the absorbance of the calibration solution with the mean

concentration (number 5) against the blank at wavelengths specified in the table below.

Pour appropriate volume of calibration solution 5 into a clean cuvette, similarly fill the

other cuvette with the blank solution. Be careful inserting the cuvettes into the

spectrophotometer not to spill the content!

Wavelength [nm] 400 450 500 550 600 650 700

Absorbance

Select the optimal wavelength for making the further measurements. Optimal wavelength

is the wavelength with the highest absorbance.

Optimal wavelength [nm]

C) Construct the calibration curve

Before plotting the calibration curve, you have to calculate the concentration of individual

calibration solutions from known dilution of the stock solution.

On a spectrophotometer, measure the absorbances of all the calibration solutions against

the blank at the optimal wavelength found in previous step.

Number Concentration Absorbance

- mmol/l -

0 0.0 0.000

1

2

3

4

5

6

7

8

9

10

According to the Beer-Lambert law, the absorbance is directly proportional to the

concentration. Therefore the calibration curve should be a straight line passing through the

origin, i.e. the point (0,0).

There is a computer in the students' laboratory with a MS Excel file prepared to fit a linear

regression line to the collected data. Before putting data into this MS Excel form, please

finish the measurement of unknown samples (next step).

8

D) Determination of concentration of unknown samples

On a spectrophotometer, measure the absorbances of all unknown sample solutions

against the blank at the optimal wavelength.

Sample Absorbance Concentration

- mmol/l

1

2

3

4

The regression equation for calibration curve generated in the previous step will be

automatically used to calculate concentrations of unknown samples.

Print the filled MS Excel form and attach it to your lab report.

Conclusion:

9

2) Identification of acid-base indicator by absorption spectra

Absorption spectrum is a graph of absorbance as a function of wavelength of light (electromagnetic

radiation). In UV-VIS range of radiation, absorption spectrum looks like a continuous curve with one or only

few maxima, whose position is characteristic for particular substance. Absorption spectra can be used for

identifying substances or for checking their purity.

Procedure:

Acid-base indicators are mostly organic dyes, which can be protonated (change ionization) according to pH

of a solution into which they are introduced. A change in ionization is coupled with a change in structure and

coloration. Acidic form and basic form of an indicator are different in colour. For identifying of an unknown

acid-base indicator, we will measure absorption spectra of both acidic and basic form.

You will get a test tube with an unknown acid-base indicator to identify. It is an aqueous

solution at pH more-less neutral (pH~7).

What is the colour of the indicator at neutral pH?

Take 2 clean test tubes. Mark one "A" (acidic) and the other one "B" (basic). Measure 1

ml of the indicator solution into both test tubes and then add 1 ml of sulfuric acid (0.05

mol/l) to "A" and 1 ml of sodium tetraborate (0.05 mol/l) to "B".

What is the colour of the acidic form?

What is the colour of the basic form?

On a spectrophotometer, measure the absorption spectra within the range 400 - 700 nm

using the interval of 10 nm of both acidic and basic forms of the indicator against distilled

water as a blank.

There is a computer in the students' laboratory with a MS Excel file prepared to put the

collected data and plot the graphs.

Print the filled MS Excel form and attach it to your lab report.

Compare the spectra with standards (from a catalogue available in the students'

laboratory) and identify the indicator.

10

Wavelength

[nm]

Absorbance

(acidic form)

Absorbance

(basic form) Wavelength

[nm]

Absorbance

(acidic form)

Absorbance

(basic form)

400

560

410

570

420

580

430

590

440

600

450

610

460

620

470

630

480

640

490

650

500

660

510

670

520

680

530

690

540

700

550

Wavelength of the absorption maximum of the acidic form:

Wavelength of the absorption maximum of the basic form:

Name of unknown acid-base indicator:

Try to find somewhere the structural formula:

11

3) Spectrophotometric estimation of Cl- concentration

Reagent for estimation of Cl- concentration contains Hg(SCN)2 and Fe(NO3)3. Cl- reacts with Hg(SCN)2

when the colorless complex [HgCl2] is formed. Free ions SCN- yields with Fe3+ red complex suitable for the

photometric determination.

Procedure:

You have prepared 3 small dry test tubes into a test tube rack. Mark them with sa

(sample), st (standard) and 0 (blank). Pipette according to the table blood serum,

standard and distilled water (on the bottom of test tube, the solution mustn´t stay

inside the tip!):

Test tube 1 (sample) 20 µl blood serum

Test tube 2 (standard) 20 µl standard Cl- (cst = 100 mmol/l)

Test tube 3 (blank) 20 µl distilled water

Pipette 2.0 ml of reagent into all test tubes. Mix the content properly.

Mix and after 5 minutes measure the absorbance of sample (Asa) and standard (Ast)

against blank at 450 nm.

Absorbance

Sample

Standard

The concentration of Cl- calculate according to: x cstandard

Csample = mmol/l

12

Chromatography

1) Paper chromatography of amino acids

1. Take a sheet of chromatographic paper (18 x 18 cm). Find out a centre of the sheet. Draw

a circle around the centre about 3 cm in diameter. You can use prepared template. This is

the "base-line", the start position. On the base-line make 6 marks evenly spaced and

number them 1-6.

Chromatogram

2. Using labelled capillaries make small spots (less than 0.5 cm in diameter) of about 5 μl of

the standards and your unknown sample. The application should be repeated 2 times.

Before the further application, the wet spots must be dried under the infra-red lamp or

a ventilator.

3. Use your pencil to perforate the paper exactly in the centre to draw through the hole

a paper "wick". Place the paper into a Petri dish partly filled with a solvent mixture

(n-butanol, acetic acid, water – 4:1:1) and cover the paper with a cap. Do not take the

Petri dish out from the fume chamber! All following procedures must be done in a fume

chamber!

Petri dish with solvent chromatogram paper wick

13

4. The chromatograms are left to develop until the head of a solvent overlaps the Petri dish

cap. The paper is removed, dried, and sprayed with ninhydrine reagent. Heat the paper at

80°C in order to develop the formation of blue complexes.

5. Calculate the individual Rf according to the equation.

Site Amino acid Rf

1 Lysine

2 Glycine

3 Alanine

4 Isoleucine

5 The mixture of 1-4 - - -

6 Unknown sample

Sample identification:

14

2) Separation of plant pigments by thin-layer chromatography

There are a few lipophilic pigments in green parts of plants. They have different chemical and

functional properties. The primary function of pigments in plants is photosynthesis which

uses the green pigment chlorophyll along with several red and yellow pigments. These

pigments can be separated using Thin Layer Chromatography (TLC).

Procedure:

1. There are prepared samples of green leaves or needles on the working place. Cut it in

pieces in a mortar. Then add a small spoon of calcium carbonate, a little bit of glass

shards and crush it with a pestle in the presence of 1-2 ml of acetone. Protect your

eyes using glasses.

2. Take two TLC plates. After the separation, one is for you to be attached to the

protocol, the second will be used further and destroyed by procedure of extraction of

one of the pigments.

3. Draw the base-line about 2 cm from the shorter edge of the plate. Use the thin pencil.

4. Dip the glass capillary into the acetone extract and touch the tip of the capillary to the

plate at the position you’ve marked. Gently and quickly make spot according to the

picture. Try to make as small spots as possible and avoid scratching of the thin layer.

For higher concentration repeat the process of application for several times.

5. Prepared separation chamber should be kept close. There is approximately 1cm thick

layer of liquid solvent mixture on the bottom and the rest volume is filled with its

vapors.

6. Place the thin layer plate carefully in the chamber (chromatography tank), containing a

solvent at the bottom. Cover the vessel with a glass plate and allow to separate about

15 min. The base – line must be situated above the level of a solvent.

15

7. When the front of a solvent almost overlaps the top of the TLC plate, remove the plate.

Evaluate the dry plate for the presence of pigments and enclose to laboratory protocol.

16

Measurement of absorption spectrum of one of the pigments

After separation of the plant pigments, take one of the TLC plates and select one of the green

strips. Use the scissors to cut out the strip from the TLC plate. Put the piece with the strip into

a test tube and add 2 ml of ethanol. Wait until complete discoloration of TLC plate piece.

Measure absorption spectrum of this fraction using a spectrophotometer in a range of

wavelengths from 350 to 700 nm. As a blank solution, use distilled water. By comparing the

spectrum with spectra of individual pigments, determine whether the fraction contains

chlorophyll a or chlorophyll b.

wavelength

nm A

wavelength

nm A

wavelength

nm A

350 470 590

360 480 600

370 490 610

380 500 620

390 510 630

400 520 640

410 530 650

420 540 660

430 550 670

440 560 680

450 570 690

460 580 700

Name of the pigment

17

3) Separation of dye mixture by gel chromatography

Task: Separate dextran blue and potassium chromate.

Dextran blue

What are dextrans?

Colour Molar mass

~ 2 × 106 g/mol

Potassium chromate

formula:

Colour Molar mass

g/mol

There is a chromatographic column filled with Sephadex gel on the working place. Sephadex

is polysaccharide of dextran type.

Procedure:

(1) Uncap the chromatographic column, open the tap and allow carefully run out the

excess of fluid until the surface of the gel bed is reached. Pipette on the gel slowly 0.5

ml of the coloured mixture (potassium chromate and dextran blue). Use the pipette

with fixed volume (=500 µl) and proper tip (blue). Pipette carefully! Not to whirl the

surface of the gel!

Colour of the mixture:

(2) By turning the tap let the sample pass into the column.

(3) Pipette (using glass pipette and rubber suction bulb) about 3 ml of elution solution

(physiologic solution; 0.9% NaCl) and let it penetrate the gel in the same way.

(4) Elute the column with sufficient amount of elution solution until both fractions leave

the outflow.

(5) Collect both fractions in calibration test tubes.

18

What is happening in the columne? Describe the changes:

Fraction

order Colour Substance Fraction volume

1

2

Explain the order of separated dyes:

19

Potentiometry

1) Potentiometric measurement of pH

Determination of pH can be done by simple colorimetric methods using acid-base indicators

(pH test strips). Nevertheless, the precision of such methods is mostly insufficient. For exact

pH measurement, laboratories are equipped with pH meters with usual resolution of 0.01 pH

units, high-end instruments with resolution of 0.001 pH units.

There is a pH meter with combined electrode (consisting of both the glass and the reference

electrode) at your working place. Electrode must be kept moist using the storage solution.

Remove the protective plastic cover with storage solution before using the electrode. Keep the

storage solution inside the plastic cover, do not pour it out. At the end of your work, you have

to place the electrode back into the cover with storage solution. Never touch the glass

membrane of the electrode with your fingers! Electrodes should be rinsed between samples

with distilled water. After rinsing, gently blot the electrode with cotton paper piece to remove

excess water.

Procedure:

Into a clean small beaker, transfer the sample which pH you want to determine, by

pouring from the small plastic bottle.

Estimate the pH using universal indicator strip.

Dip the electrode of the pH meter into the solution in the beaker, gently shake the content

by slowly moving the beaker to make the membrane of the electrod be in contact with the

solution. (Electrode is "dirty" with distilled water used for rinsing between samples.)

After stabilization of the value, read the pH on a display.

Put the sample back into the small plastic bottle.

Sample pH estimated by indicator strip pH measured by pH meter

20

2) Demonstration of buffer functioning

Buffers are solutions that maintain a relatively stable pH and resist changes in pH, upon

addition of small amounts of acid or base. At your working place, there are two bottles with

solutions to compare how they are resistant to changes in pH. One bottle contains unbuffered

solution - deionized (ultrapure) water (in theory, pH=7), the other one contains phosphate

buffer at pH close to 7.

Unbuffered solution - water

Using a graduated cylinder, measure 50 ml of deionized water, pour it into a clean beaker and

determine the pH by pH meter. Record the value (measurement 1). Pour out the water from

the beaker. Using the same graduated cylinder, measure again 50 ml of deionized water, pour

it into the same beaker and determine the pH by pH meter (measurement 2). After the second

measurement, do not remove the combined electrode from the beaker, let it dipped there. By

pipetting add 100 μl of HCl solution (c=0.1 mol/l) into the solution in the beaker and gently

mix the content. After stabilization of the value record the pH.

pH of deionized water pH after addition of HCl

measurement 1

measurement 2

Describe how stable is the pH of an unbuffered solution:

Buffer

Using a graduated cylinder, measure 50 ml of phosphate buffer, pour it into a clean beaker

and determine the pH by pH meter. Record the value (measurement 1). Pour out the buffer

from the beaker. Using the same graduated cylinder, measure again 50 ml of phosphate

buffer, pour it into the same beaker and determine the pH by pH meter (measurement 2).

After the second measurement, do not remove the combined electrode from the beaker, let it

dipped there. By pipetting add 100 μl of HCl solution (c=0.1 mol/l) into the solution in the

beaker and gently mix the content. After stabilization of the value record the pH.

pH of the buffer pH after addition of HCl

measurement 1

measurement 2

Describe how stable is the pH of a buffer solution:

Describe the differences between the behaviour of the buffer solution, and the pure water,

upon the addition of a small amount of a strong acid:

21

3) Potentiometric titration

Potentiometric titration is a volumetric method in which the potential between two electrodes

is measured as a function of the added reagent volume. In potentiometric titrations, there is no

need for indicator. The titration is not stopped at the equivalence point, the whole titration

curve is constructed instead. Volume of the standard reagent consumed to reach the

equivalence point is found by analysis of the titration curve. A titration curve has a

characteristic sigmoid shape. The part of the curve that has the maximum change marks the

equivalence point of the titration.

You will be performing two potentiometric titrations to determine the unknown

concentrations of two acids. In order to perform these titrations you will utilize a pH meter.

A) Strong acid: hydrochloric acid (HCl)

B) Weak acid: acetic acid (CH3COOH)

A) Titration of a strong acid, graphical analysis of the titration curve

Procedure:

Into a clean small beaker, using a glass pipette measure exactly 10.0 ml of the sample

(strong acid – HCl) which concentration you want to determine. Add a small stirring bar

(little white corpuscle similar to Tic Tac mints) into the beaker.

Close the burette tap, and fill the burette with the standard solution of NaOH (c=0.100

mol/l) until the meniscus is about 1-2 cm above the zero mark. Remove the funnel. Open

the tap and allow the solution to drain (into the waste bottle) until the meniscus falls to

zero mark. Fill the burette with the standard solution. The burette is ready for the titration.

Set up the apparatus so that you can perform the potentiomeric titration. Place the beaker

with the sample on a magnetic stirrer. Dip the electrode of the pH meter into the solution

in the beaker. If the electrode is not sufficiently submerged, add a little of distilled water.

Place the burette so that the orifice is slightly above the beaker and is possible to use it for

adding standard solution to the sample. Switch on the stirring (not heating!) and adjust the

speed of it to optimal (not extremely fast to prevent damage of the electrode).

Before adding any NaOH from the burette, record the initial pH on the display of the

pH meter. Put the data into the table further in this instruction sheet. You are now ready to

begin the titration. You will be adding NaOH solution in increments 0.5 ml until 10.0 ml.

After every addition of NAOH, record the pH. Be sure that the pH has stabilized! There is

a delay between the addition of NaOH and a stable pH reading.

There is a computer in the students' laboratory with a MS Excel file prepared to put the

collected data and plot the graph (titration curve).

Print the filled MS Excel form.

Use a graphical method to find out the equivalence point.

Two parallel lines are drawn tangent to the flat

portions of the curve. A line perpendicular to both the upper and the

lower tangent is drawn to help you find the middle between them so that

you can plot the third parallel line exactly in the middle. The point where

this middle parallel crosses the titration curve is the equivalence point.

22

Potentiometric titration of a strong acid (HCl) – data sheet

Added NaOH volume (ml) pH

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

HCl

Consumption of NaOH read from titration curve:

Calculation: (M = 36.5 g/mol)

Molar (substance) concentration c = mmol/l

Mass concentration = g/l

23

B) Titration of a weak acid, graphical + methematical analysis of the titration curve

Procedure: (the same as for strong acid)

Into a clean small beaker, using a glass pipette measure exactly 10.0 ml of the sample

(weak acid – CH3COOH) which concentration you want to determine. Add a small

stirring bar (little white corpuscle similar to Tic Tac mints) into the beaker.

Close the burette tap, and fill the burette with the standard solution of NaOH (c=0.100

mol/l) until the meniscus is about 1-2 cm above the zero mark. Remove the funnel. Open

the tap and allow the solution to drain (into the waste bottle) until the meniscus falls to

zero mark. Fill the burette with the standard solution. The burette is ready for the titration.

Set up the apparatus so that you can perform the potentiomeric titration. Place the beaker

with the sample on a magnetic stirrer. Dip the electrode of the pH meter into the solution

in the beaker. If the electrode is not sufficiently submerged, add a little of distilled water.

Place the burette so that the orifice is slightly above the beaker and is possible to use it for

adding standard solution to the sample. Switch on the stirring (not heating!) and adjust the

speed of it to optimal (not extremely fast to prevent damage of the electrode).

Before adding any NaOH from the burette, record the initial pH on the display of the

pH meter. Put the data into the table further in this instruction sheet. You are now ready to

begin the titration. You will be adding NaOH solution in increments 0.5 ml until 10.0 ml.

After every addition of NAOH, record the pH. Be sure that the pH has stabilized! There is

a delay between the addition of NaOH and a stable pH reading.

There is a computer in the students' laboratory with a MS Excel file prepared to put the

collected data and plot the graph (titration curve).

Print the filled MS Excel form.

Use both, a graphical method and mathematical analysis to find out the equivalence point.

Mathematical analysis of the titration curve

Consumption of the standard reagent in equivalence point (V) can be calculated using the

formula:

ΔVpHΔpHΔ

pHΔVV

22

2

V+ "added NaOH volume" at last positive 2pH

2pH+ the last positive 2pH

2pH- absolute value of the first negative 2pH

V the difference in "added NaOH volume" between the last positive and the first

negative 2pH (In our experiment, it always was 0.5 ml.)

24

Potentiometric titration of a weak acid (CH3COOH) – data sheet

Added NaOH

volume (ml) pH pH 2pH

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

pH = pHx+1 - pHx 2pH = pHx+1 - pHx

25

CH3COOH

Consumption of NaOH read by graphical method from a titration curve:

Consumption of NaOH calculated:

Calculation: (M = 60.0 g/mol)

Molar (substance) concentration c = mmol/l

Mass concentration = g/l

26

4) Determination of the isoelectric point of an amino acid

Added NaOH

volume (ml) pH

Added NaOH

volume (ml) pH

0.0

- -

0.5

10.5

1.0

11.0

1.5

11.5

2.0

12.0

2.5

12.5

3.0

13.0

3.5

13.5

4.0

14.0

4.5

14.5

5.0

15.0

5.5

15.5

6.0

16.0

6.5

16.5

7.0

17.0

7.5

17.5

8.0

18.0

8.5

18.5

9.0

19.0

9.5

19.5

10.0

20.0

Calculation of pI:

27

Osmolality, osmosis, dialysis

1) Demonstration of osmosis

We will try to carry out a classical experiment on

demonstration of osmosis. The principle is shown in

the figure. Water moves from the solution of lower

osmolality, across the semipermeable membrane,

into the solution of higher osmolality, and its level

rises. In theory, the process should stop in time of

balancing the osmotic pressure by hydrostatic

pressure of the column of the solution inside a tube.

π = h g

h ... difference in solution levels

density of the solution

g ... gravitational acceleration

1. On a wider portion of the tube, fasten the cellophane, which will serve as a semi-

permeable membrane.

2. Dip the tube membrane down into a graduated cylinder with distilled water so that the

water level in the cylinder reached the mark.

3. Into the tube, transfer carefully by pipetting saturated sucrose solution, so that liquid

levels in the tube and in the cylinder are at the same level. For better visibility, sucrose

solution was colored with blue food coloring.

4. Let the experiment run until the end of the class. Then, using a ruler, measure the

difference between the two levels and make a conclusion.

The beginning: "What time is it?"

The end: "What time is it?"

Time period:

min

Difference in solution levels (mm):

Conclusion:

28

29

2) Determination of osmolality using cryoscopy

Preparing of 250 ml of Ringer's solution

Physiological solution in form of 0.9% NaCl contains only Na+ and Cl- ions. However, there

are also other ions in the blood plasma. In clinical practice, there are more types of infusion

solutions in use, some of them more similar in ionic composition to blood plasma. Ringer's

solution is one of them.

1. Weigh stepwise using a plastic weighing boat and transfer weighed quantities into an

Erlenmayer flask (of volume of 250 ml):

sodium chloride (NaCl) 2.150 g

potassium chloride (KCl) 0.075 g

calcium chloride (CaCl2) 0.083 g

2. Into the same Erlenmayer flask, flush also unobservable remnants from the plastic

weighing bottle using a squirt bottle with distilled water.

3. Into the same Erlenmayer flask, add distilled water to a volume about 100 – 150 ml and

by swirl mixing thoroughly dissolve the contents.

4. Using a funnel pour the content of Erlenmayer flask into a 250 ml volumetric flask. Rinse

the Erlenmeyer flask at least 2x with a little of distilled water, pour everything into a

volumetric flask.

5. Fill the volumetric flask exactly to the mark with distilled water (i.e. a volume of 250 ml).

6. Close the volumetric flask by a stopper and thoroughly mix the contents.

What is the concentration of the individual ions in the solution prepared?

M(NaCl) = 58.45 g/mol

M(KCl) = 74.56 g/mol

M(CaCl2 . 2H2O) = 146.99 g/mol

Result

Na+ mmol/l

K+ mmol/l

Ca2+ mmol/l

Cl- mmol/l

From the known composition, calculate the osmolality (osmolarity) of this solution:

30

Measurement of osmolality by modern osmometer

There is a modern freezing point osmometer Osmomat 3000 in the laboratory. The total

osmolality of aqueous solutions is determined by comparative measurements of the freezing

points of pure water and of solutions. The instrument requires very small sample volumes (50

l), test time is short (60 s).

Preparing samples:

Distilled water – it is available in the laboratory

Ringer's solution – you have already prepared (previous exercise)

Ringer's solution + ethanol

Using a graduated cylinder, measure 50 ml of Ringer's solution, pour it into a small beaker

and add by pipetting 0.25 ml of 40% ethanol.

Calculate what rise of osmolality (osmolarity) should the addition of ethanol result in:

Ringer's solution + glucose

Using a graduated cylinder, measure 50 ml of Ringer's solution and pour it into a small

beaker. Weigh using a plastic weighing boat 270 mg of glucose and dissolve it in the solution

in the beaker.

Calculate what rise of osmolality (osmolarity) should the addition of glucose result in:

Sample Measured osmolality

Distilled water mmol/kg

Ringer's solution mmol/kg

Ringer's solution + ethanol mmol/kg

Ringer's solution + glucose mmol/kg

31

3) Monitoring the kinetics of dialysis

This exercise demonstrates separation of high and low molecular weight substances (starch

and NaCl) by dialysis. Moreover, we will monitor dialysis rate of chlorides. As a dialysis membrane,

we will use a cellophane with the size of pores between 3 and 4 nm. These pores are permeable to

small ions, but do not allow the passage of large molecules.

Procedure:

Preparing the dialysis experiment:

1. There is an empty dialysis container mounted the laboratory stand at your working

place. The dialysis container is a glass tube with cellophane membrane on one side.

2. Using a graduated cylinder, put 150 ml of distilled water into the beaker (of a volume

of 250 ml). Keep the dialysis container out of the beaker at this moment. Add

a stirring bar into the beaker, place the beaker on a magnetic stirrer, switch on the

stirring (not heating!) and adjust the speed of it to optimal.

3. Into the dialysis container, pipette 5 ml of starch solution and 10 ml of NaCl

solution (c = 1.5 mol/l). Prepare a stopwatch so that it can be started immediately

when you dip the dialysis container into the beaker (cellophane membrane must be

below the level of the solution in the beaker). Before starting the experiment,

ensure yourself you know what to do further (taking the samples as described

below)! Let the experiment run for 90 minutes.

Monitoring of dialysis rate:

At points in time of 2, 4, 6, 8, 10, 15, 20 minutes after starting the experiment, and

later in 10-minute intervals, take (using a pipette) from the beaker samples of a

volume of 2.5 ml. Put the samples into titration flasks labelled with numbers

corresponding to the time sample was taken.

Time (min) 2 4 6 8 10 15 20

Vtitration (ml)

Vdif (ml)

c (mmol/l)

n (mmol)

Time (min) 30 40 50 60 70 80 90

Vtitration (ml)

Vdif (ml)

c (mmol/l)

n (mmol)

Vtitration mercurimetric titration of the sample - consumption of the standard reagent

Vdif volume of the solution in the beaker (It is getting smaller by taking samples!)

c concentration of Cl- in the solution in the beaker

n amount of Cl- in the solution in the beaker

32

Estimation of chloride concentration:

Concentration of Cl- can be estimated by mercurimetric titration. Mercurimetry

belongs to complexometric titration methods. Standard reagent is a solution of Hg(NO3)2, that

forms with Cl- ions soluble non-dissociated complex [HgCl2]:

Hg2+ + 2 Cl- [HgCl2] f = n(Cl-)/n(Hg2+) = 2/1

For indication of the end point of the titration, we will use diphenylcarbazone that forms

with the excess of Hg2+ ions blue-violet coloration. The solution of Hg(NO3)2 is toxic! Keep

safety in mind! After finishing your work, put the waste into the extra bottle!

To the sample taken from the beaker you put into a titration flask, add 2.5 ml of distilled

water and 0.3 ml of indicator (diphenylcarbazone). Titrate with standard solution of Hg(NO3)2

(cstandard reagent = 12.5 mmol/l) to a color change to blue-violet coloration.

Calculate the concentration of chlorides in individual samples and the amount of chlorides

passed through the dialysis membrane into the beaker. Fill in the values into the table.

sample

titrationreagent standard

V

.f.Vcc

dif.Vcn

Vsample = 2.5 ml

Vdif = 150 ml, 147.5 ml, 145 ml, ... (It is getting smaller by 2.5 ml every time the sample is taken.)

Checking what has happened with the starch:

After taking of the last sample (90 minutes), stop stirring and take up the dialysis

container from the beaker. Take two test tubes and transfer a small volume (~ 1 ml) of the

solution from the dialysis container into one and from the beaker into the other one. Add 3

drops of Lugol solution (solution of iodine) into both test tubes and compare the results.

Lugol test - solution from the dialysis container:

Lugol test - solution from the beaker:

Conclusion:

33

Evaluation of the dialysis rate:

Make a graphical evaluation. Plot a graph showing increase in time of the amount of

chlorides in the solution in the beaker. First, plot the individual points according to the data in

the table. For to=0 the amount of chlorides in the beaker is no=0.Then draw optimal interlaced

curve.

axis x ... time (minutes)

axis y ... n = amount of chlorides in the beaker (mmol)

Divide the scale on axis x into 5-minute intervals (time t1, t2, t3, ...) and from the curve

read on axis y the corresponding amount of chlorides (n1, n2, n3, ...).

Time (min) 5 10 15 20 25 30 35 40 45

nx (mmol)

vx (mmol Cl-/min)

Time (min) 50 55 60 65 70 75 80 85 90

nx (mmol)

vx (mmol Cl-/min)

34

Dialysis velocity (v) is defined as the amount of Cl- passing through the semipermeable

membrane per minute.

v1 = (n1 - n0) : 5 [mmol Cl-/min]

v2 = (n2 - n1) : 5

v3 = (n3 - n2) : 5 etc.

Plot a graph of dialysis velocity in time.

axis x ... time (minutes)

axis y ... v = calculated velocity (mmol Cl-/min)

At the cross section of the curve with axis x, read the time point at which dialysis stopped due

to loss of concentration gradient (balancing of Cl- concentrations on both sides of the

membrane). By extrapolation of the curve to zero time, find out the initial velocity at the cross

section of the curve with axis y.

Time point at which dialysis stopped:

Initial velocity:

Conclusion:

35

Antioxidants

Processes happening in human body may produce reactive oxygen species (ROS) and

reactive nitrogen species (RNS), collectively called RONS (Reactive Oxygen and Nitrogen

Species). These RONS are highly reactive and comprise compounds of both radical and non-

radical nature.

Reactive oxygen species (ROS)

Radicals Non-radicals

superoxide anion (superoxide) O2•- hydrogen peroxide H2O2

hydroperoxyl radical HO2• hypochlorous acid HClO

hydroxyl radical HO• ozone O3

peroxyl radicals ROO• singlet oxygen 1O2

alkoxyl radicals RO•

Reactive nitrogen species (RNS)

Radicals Non-radicals

nitric oxide NO• nitrous acid HNO2

nitrogen dioxide NO2• dinitrogen trioxide N2O3

peroxynitrite ONOO-

alkyl peroxynitrite RONOO-

Free radicals can be defined as molecules, atoms or ions, capable of independent existence

that contain et least one unpaired electron. Free radicals may be created in a number of

ways:

1) homolytic bond cleavage

2) single-electron reduction, i.e. by adding one electron

3) single-electron oxidation, i.e. by losing one electron

Chemical reactions involving free radicals are usually chain reactions with an initiation,

a propagation and a termination step. Because of the propagation phase of the chain reaction,

free radicals have the potential to be extremely harmful to biomolecules (including DNA,

proteins, and the lipids of cell membranes). Once a free radical is generated, it can react with

stable molecules to form new free radicals from them. These new free radicals go on to

generate new free radicals, and so on.

It is not surprising that living organisms are equipped with many protection systems against

free radicals. We can distinguish:

1) enzymatic systems (e.g. superoxide dismutase, catalase, glutathione peroxidase)

2) low molecular weight antioxidants (also known as 'free radical scavengers')

A free radical scavenger is a molecule that reacts with a high energy free radical species in a

propagation step of a chain reaction, forming a more stable radical species which can be

eliminated in some way before further damage is done to biomolecules.

36

Measurement of total antioxidant capacity (TAC)

Total antioxidant capacity (TAC) describes overall abilility of the sample to resist to

oxidative stress. There are several methods for the measurement of total antioxidant capacity,

different methods may evaluate it in a different way. We will use two different methods:

A) TEAC (Trolox Equivalent Antioxidant Capacity)

This method measures antioxidant capacity of the sample, as compared to the standard

- Trolox (synthetic water-soluble analogue of vitamin E). The principle of the method is

a "scavenging" (= inactivation, decomposition) of an artificially prepared stable radical ABTS

[2,2'-azino-bis(3- 3-ethylbenzothiazoline-6-sulphonic acid)]. ABTS is converted to its radical

cation ABTS+ by addition of sodium persulfate (peroxydisulfate). This radical cation is blue

in color and absorbs light at 734 nm. Samples with antioxidant activity convert the blue

ABTS+ radical cation back to its colorless non-radical form. The reaction may be monitored

spectrophotometrically at the wavelength 734 nm.

Trolox (analogue of vitamin E)

Formation of ABTS+ radical by the action of persulfate; reaction of the ABTS+ radical with antioxidant (AOH)

B) FRAP (Ferric Reducing Antioxidant Power)

This method describes total reducing activity of the analysed sample. The principle is

a reduction of ferric complexes Fe3+-TPTZ, which are colourless, into purple ferrous Fe2+-

TPTZ products. The change in colour may be monitored spectrophotometrically at the

wavelength 593 nm. The result is given as an equivalent concentration of Fe2+ ions.

TPTZ (2,4,6-tri(2-pyridyl)-1,3,5-triazine)

37

Exercise 1: Making calibration curves

Calibration curve for TEAC method was prepared by the lab assistants, you can find it (a

plot) at your working place. You have to measure yourself just the calibration curve for

FRAP method.

Prepare calibration curv fore FRAP method (Ferric Reducing Antioxidant Power)

1) Using stock solution of iron (II) sulfate (cFeSO4 = 1 mmol/l), prepare a series of

standard solutions with different concentrations in small plastic test tubes. Calculate

the concentrations of these calibration solutions.

2) Pipette 2 ml of FRAP reagent into numbered test tubes. Concerning the standard

solutions, in all cases add 50 l in precisely same time periods (recommendation: 30

s). Exactly in 10 minutes after addition , measure the absorbance against blank (test

tube 0) at the wavelength 593 nm. (In time of addition of 50 l from test tube 0, start

the stopwatch and keep it running. After 30 s, add 50 l of standard solution 1 into the

test tube 1, and so on. Exactly at 10 minutes after starting stopwatch, set the zero

absorbance using the blank solution, at 10 minutes 30 s make the measurement of the

absorbance of standard 1, and so on.)

test tube number 0 1 2 3 4 5 6 7 8 9 10

stock sol. of FeSO4 (l) 0 50 100 150 200 250 300 350 400 450 500

distilled water (l) 500 450 400 350 300 250 200 150 100 50 0

cFeSO4 (mmol/l) 0

A593 0

3) Plot calibration graph A593 = function of cFeSO4 axis y … A593, axis x … cFeSO4

38

Exercise 2: Measurement of total antioxidant capacity of different samples

You will measure total antioxidant capacity of three different samples by two methods

(TEAC and FRAP). First, you have to prepare the samples:

sample 1 – blood serum – it is ready to use at your working place

sample 2 – saliva

Do not eat or drink during the 30 minutes preceding collection. Open the Salivette tube

and put the cotton roll into your mouth. Gently chew the cotton roll and swirl it around the

mouth for about 1 minute until the cotton is saturated in saliva. Place the saturated cotton

roll back inside Salivette tube and close the lid. Centrifuge at 2.500 RPM for 2 minutes.

sample 3 – commercially available antioxidant supplement ("miraculous pill")

In a mortar, crush a pill of antioxidant supplement. Transfer a powder to a small beaker

and dissolve it in 5 ml of distilled water. Prepare filtration apparatus (funnel with filtrate

paper) and and use it to remove insoluble deposits. Transfer 0.5 ml of the filtrate in a

beaker and dilute it with 100 ml of distilled water. This diluted solution of antioxidant

supplement is the sample 3.

A) TEAC method

Pipette 2 ml of ABTS reagent into numbered test tubes. Concerning the samples, in all cases

add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in 10 minutes

after addition of the sample, measure the absorbance against blank (test tube 0) at the

wavelength 734 nm. Using the calibration curve for TEAC method, read the corresponding

values of trolox concentration.

test tube number - sample 0 1 - serum 2 - saliva 3 - pill

ABTS reagent (l) 2000 2000 2000 2000

sample (l) - 50 50 50

distilled water (l) 50 - -

A734 0

cTROLOX (mmol/l) 0

B) FRAP method

Pipette 2 ml of FRAP reagent into numbered test tubes. Concerning the samples, in all cases

add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in 10 minutes

after addition of the sample, measure the absorbance against blank (test tube 0) at the

wavelength 593 nm. Using the calibration curve for FRAP method, read the corresponding

values of FeSO4 concentration.

test tube number - sample 0 1 - serum 2 - saliva 3 - pill

FRAP reagent (l) 2000 2000 2000 2000

sample (l) - 50 50 50

distilled water (l) 50 - -

A593 0

cFeSO4 (mmol/l) 0

39

Exercise 3: Measurement of total antioxidant capacity of vitamin C solutions

Using stock solution of vitamin C (c=1 mmol/l), prepare a series of solutions with different

concentrations in small plastic test tubes:

test tube number 0 1 2 3 4 5

stock sol. of vit. C (l) 0 100 200 300 400 500

distilled water (l) 1000 900 800 700 600 500

cvitamin C (mmol/l) 0

A) TEAC method

Pipette 2 ml of ABTS reagent into numbered test tubes. Concerning the samples of vitamin C,

in all cases add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in

10 minutes after addition of the sample, measure the absorbance against blank (test tube 0) at

the wavelength 734 nm. Using the calibration curve for TEAC method, read the

corresponding values of trolox concentration.

test tube number 0 1 2 3 4 5

ABTS reagent (l) 2000 2000 2000 2000 2000 2000

vit. C sample (l) 50 50 50 50 50 50

A734 0

cTROLOX (mmol/l) 0

B) FRAP method

Pipette 2 ml of FRAP reagent into numbered test tubes. Concerning the samples of vitamin C,

in all cases add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in

10 minutes after addition of the sample, measure the absorbance against blank (test tube 0) at

the wavelength 593 nm. Using the calibration curve for FRAP method, read the

corresponding values of FeSO4 concentration.

test tube number 0 1 2 3 4 5

FRAP reagent (l) 2000 2000 2000 2000 2000 2000

vit. C sample (l) 50 50 50 50 50 50

A593 0

cFeSO4 (mmol/l) 0

40

Enzymology I

1) Enzyme specificity

Introduction:

Most of the enzymes are highly specific for the reaction to be catalyzed and for the

substrate to be selected. This property will be demonstrated in reaction with adequate

substrate. We shall demonstrate also the thermolability of enzymes. We shall use α-amylase

from saliva and sucrase of baker´s yeast (Saccharomyces cerevisiae). Sucrase hydrolyses β-

glycosidic bond of sucrose to glucose and fructose. This enzyme is different from sucrose in

the intestinal juice of mammals which hydrolyses α-1 glycosidic bond.

Procedure:

1. Take approximately 0.5 g of yeast and suspend in 2 ml of distilled water. This is the

sucrase solution.

2. Take diluted saliva from previous experiment as amylase solution.

3. Prepare a set of 6 test tubes and pipette according to the table. Perform the

described experiment:

Test tube number 1 2 3 4 5 6

Amylase (ml) 0.5 0.5 0.5

Sucrase (ml) 0.5 0.5 0.5

Boil (ml) yes yes

Starch (ml) 2.0 2.0 2.0

Sucrose (ml) 2.0 2.0 2.0

Place into the water bath (37°C) for 30 minutes. Then divide the content of each tube into two parts. The first portion will be examined with Fehling reagent (mix 2 ml of Fehling I + 2 ml of Fehling II, add 0.5 ml of this solution to each tube and boil it) and second with iodine (1 drop of iodine solution + 5 ml of water). Recorde the result:

With Fehling r.

With iodine

41

4. Evaluate the result in each test tube and make conclusions in your protocol:

Tube No

Explanation

1

2

3

4

5

6

42

2) Dependence of activity of α-amylase on pH

Introduction:

Most of the enzymes are strongly pH-dependent in their activity. This will be

demonstrated on α-amylase which is present in the saliva of oral cavity and in the pancreatic

juice. It hydrolyses starch and glycogen to maltose, maltotriose, and a mixture of branched

oligosaccharides, and non-branched oligosaccharides. At pH 4 the enzyme is completely

inactivated.

Procedure:

1. Prepare a set of 7 test tubes and mark them. Pipette the buffers according to the

table:

Test tube Na2HPO4 (ml) Citric acid (ml) pH Colour

1 2.9 2.1 5.6

2 3.1 1.9 6.0

3 3.5 1.5 6.4

4 3.8 1.2 6.8

5 4.3 0.7 7.2

6 4.7 0.3 7.6

7 4.9 0.1 7.8

2. Add 1 ml of 1% starch solution (not containing buffer!) into each tube as substrate.

3. About 0.5 ml of saliva dilute with 6 ml of water (not buffer). Add 0.5 ml of diluted

enzyme solution to each tube and leave in water bath (37°C) for 10 minutes.

4. After 10 minutes of incubation pour approximately half of the reaction mixture into

another set of tubes and the remaining half leave on the water bath for another 5

minutes.

5. To each of the samples taken of the incubation add 5 ml of water and 1 drop of

iodine solution (Lugol). Register the colour that develops.

6. After the second incubation is completed take the samples of the bath, add again 5

ml of water to each tube and again 1 drop of iodine. Register the colour again.

7. Evaluate the results:

What pH is the most suitable for α-amylase?

43

3) Estimation of alpha-amylase in urine

a) by the test – BIOSYSTEMS – EPS

Principle of the method:

Amylase catalyzes the hydrolysis of synthetic substrate (4- nitrophenyl-

maltoheptaoside-ethylidene) to smaller oligosacharides which are hydrolyzed by α-amylase

liberating 4- nitrophenol. The amount of arising 4-nitrophenol correspond with the catalytic

concentration of alpha-amylase.

Procedure:

1. Switch on photometer ECOM E 6125 and the printer. Deprres the key ABSORBANCE

and with arrow keys set the wavelength to 405nm. Rinse several times the cuvette

in phofometr with distilled water. The cuvette is emptied with the pump.

(Ask the lab assistant for the demonstration how to use it.)

2. Stop the pump and fill the cuvette with distilled water as blank. Check the display of

the photometr. It should show ABSORBANCE 405 nm BLANK. If not, used the key

BLANK to obtain this parameter. Depress the key ENTER- MEASURE, the absorbance

on the display should be 0,000.

3. Sample measurement: Mix into Eppendorf tube - 20µl of urine and 1000µl of the

“Reagent solution “, immediately transfer into cuvette and start the stop-watch.

Measure the absorbance of sample exactly after 2nd, 3rd, 4th and 5th minute. The

measurement is achieved by depressing the key ENTER-MEASURE. The result of

measurement is always printed out.

4. Rinse several times the cuvette with distilled water and switch of the pump,

photometer and printer.

5. From the values obtained, calculate the average ΔA/min. and substituting in formula

concentration α-amylase in the urine sample.

Calculation: Urine measurements at 37⁰C (reaction temperature) ∆A1= A3 – A2 ∆A2= A4 – A3 ∆A3= A5 – A4 mathematical average ∆A /min = ∆A1+ ∆A2+ ∆A3 / 3

µkat/l = ∆A /min x 105,9 (conversion factor)

U/L = ∆A /min x 6384 (conversion factor)

Benchmarks at 37⁰C µkat/l = 0,26-8,15 U/L = 16 - 491

44

b) Wohlgemuth´s test

Procedure:

1. Into a series of ten test tubes pipette 1 ml of 0.9% NaCl with the exception of the

first test tube.

2. Into tube No 1 and 2 place 1 ml of urine.

3. Mix the content of tube 2 and take over 1 ml to the third test tube. Mix, measure

off again 1 ml and in this way continue the dilution of urine in the following test

tube. The last 1 ml volume from tube No 10 should be discarded.

45

4. To each test tube add 2 ml of the buffered 0.1% starch solution. Allow incubate in

a water bath at 45°C for 15 min.

5. Cool test tubes and add into each tube 3 drops of iodine solution. Examine the

coloration of the content. Record the number of the last test tube which gives no

blue colour with iodine.

6. Calculate the activity in Wohlgemuth units corresponding the volume (in ml) of

0.1% starch digested by 1 ml of urine under conditions described.

For example: if ¼ ml of urine (dilution 4x) digested 2.0 ml of starch, the enzymic activity equals 2 x 4 = 8 U. The Wohlgemuth units correspond very roughly to µkalt/l. Complete urine dilution:

1x 2x

Conclusion:

46

Enzymology II

1) Estimation of Michaelis' constant of acid phosphatase

Principle:

Phosphatases cleav esters bonds of phosphoric acid and phosphate and a relevant alcohol are

arised. A synthetic substrate which is possible for a direct photometric estimation is used for

observation the enzyme activity.

The reaction will be done with different substrate concentration and the relation of the

reaction velocity and substrate concentration will be observed. Graphic expression will be in

system of reciprocal values.

Solutions: citrate buffer pH 4,96 (approximately 0.01 mol/l)

substrate solution (4-nitrophenolate, sodium salt 2.5 mmol/l in citrate bufer)

inhibition solution (0.1 mol/l of NaOH – 0.03 mol/l EDTA)

emzyme solution (acid phosphatase in Eppendorf)

Procedure:

Read the whole procedure before you start working!

To obtain good results it is necessary to pipette exactly and to keep the time of incubation!

1. Prepare 10 test tubes and mark them.

2. Pipette according to the table citrate buffer at first and then substrate (use the

mechanical micropipettes, it is possible to use the same tip, after the finishing the

work waste the tip).

Test tube No. 1 2 3 4 5 6 7 8 9 10

Citrate buffer [l] 450 400 350 300 250 200 150 100 50

Substrate [l] 50 100 150 200 250 300 350 400 450 500

3. Place the set of test tubes into the water bath at 37C for 5 minut. Meanwhile, bring

the enzyme from the refrigerator, prepare stop watch and the pipette of 50 l with a

clean tip.

4. Enzyme solution must be pipetted in absolutely exact 20 seconds period. Add 50 l of

the enzyme to the test tube No.1, press the stop watch and let it run during the whole

experiment. Than pipette the same volume of the enzyme solution to other test tubes

in 20 seconds period. Just take out the test tube from the water bath and after adding

the enzyme solution place it back immediately. Put the pipette tip approximately 0,5

47

cm above the level, it is not advisible to touch the substrate. It is necessary to drop the

enzyme solution in the substrate not on the wall of the tube.

5. Prepare the second test tube and add the enzyme after 20 seconds exactly. Continue in

the same way, the period of adding into tubes must be the same, 20 seconds!!!

6. Prepare the inhibition solution which stops the enzyme reaction. The inhibitor must be

added in 10 minutes after adding the enzyme, it means the reaction runs for 10

minutes in each test tube. Add 2 ml of inhibition solution to each test tube and mix.

Do not place back the tubes to the water bath than switch off the stop watch.

7. Measure the absorbance of all samples at 405nm against water (blank). Start with tube

No. 1 and continue in sequence to tube No. 10. Do not rinsen the cuvettes, only pour

them and dry them carefully.

8. Enter the obtained values into the table in the computer. The concentration of basic

substrate solution is 2.5 mmol/l. Measured absorbance is proportional to the enzymatic

reaction velocity in each tube because reaction velocity means amount of converted

substrate in time. In this experiment the result is amount of the arisen product in 10

minutes. The program creats Lineweaver-Burk`s graph, it is dependence of 1/v on

1/[S] and the equation for the line is given.

Calculation:

Michaelis` constant is estimated from the equation or from the graph where the value of -1/KM

is found.

Elaborate a report and add the table and the graph.

48

2) Estimation of catalase activity

Principle:

Catalase of erythrocytes is a part of a system reducing unadvisable reactive forms of oxygen,

more precisely their products. Catalase released from erythrocytes decomposes hydrogen

peroxide. Concentration of substrate, i.e. hydrogen peroxide, is determined by manganometric

titration.

Solutions: substrate solution (0.01 mol/l of hydrogen peroxide in 0.1 mol/l phosphate

buffer pH 6,8)

standard solution of potassium permanganate KMnO4 (0.32 g KMnO4 in 1l)

sulfuric acid (1 mol/l)

enzyme solution (hemolyzed red blood cells 100x diluted)

Procedure:

1. Prepare 10 titration flasks, mark them numbers 0-9. Put 2.5 ml of sulfuric acid into

each flask (it will stop the enzymatic reaction and it is also necessary for the

manganometric titration).

2. Setting up enzymatic reaction:

Measure 60 ml of substrate into 100 ml Erlenmayer flask (buffered hydrogen

peroxide solution) and add a magnetic stirrin bar. Place the flask on a magnetic stirrer.

3. Prepare 5 ml pipete for a sampling and transfer 5 ml of substrate into the titration

flask number 0. Be careful, the pipete must not be contaminated with sulfuric acid or

else it could not be used for another sampling.

4. Measure 1 ml of enzyme solution (hemolyzed red blood cells 100x diluted) and add it

to the substrate and run the stop watch immediately and let it run.

5. Prepare 5 ml pipete for a sampling in 1 minute intervals and transfer samples into

titration flasks immediately. The moment of sample releasing into the titration flask

with sulfuric acid that stops the enzymatic reaction is decisive. It means, suck in the

sample several seconds before the minute and empty the pipete in a specific moment

exactly. All samples are titrated with the solution of permanganate and the

concentration of substrate (hydrogen peroxide) in each sample is determined.

6. Repeat the experiment with 10 times diluted enzyme solution, i.e. overall dilution is

1000x (1 ml of enzyme solution + 9 ml of water, or add just 0.1ml of enzyme solution

to the substrate).

49

7. Values obtained from the titration put in the Excel table in the computer and the

expression of changes of substrate concentration and changes of reaction velocity on

time.

8. Assess the kinetics and try to estimate a reaction degree.

9. Print the graph and enclose it to your report.

50

Enzymology III

1) Estimation of lactate dehydrogenase activity in blood serum

Principle:

Lactate dehydrogenase catalyses the reversible interconversion of pyruvate to lactate with

oxidation of NADH + H+ to NAD+. The rate of absorbance decreses of NADH at 340nm is

used for kinetic estimation of enzyme activity.

Solutions: „working solution“ - 4 ml of R1 (tris 127 mmol/l, pH 7,2, NaCl 323 mmol/l,

pyruvate 2,5 mmol/l) a 1 ml of R2 (NADH 6,6 mmol/l)

standard serum

examinate serum

Procedure:

1. Switch on photometer Libra S 6+ (heating at 37˚C) and adjust the wavelength 340nm.

2. Put a glass cuvette for measuring LD into the photometer.

3. Pipette 1000 μl of destilled water (blank) and measure A = 0.000.

4. Ampty a glass cuvette and put it back into the photometer.

5. Estimation of standard activity:

Pipette 20 μl of standard and 1000 μl of „working solution“ ,start up stopwatch, mix

the solution and insert it into the cuvette immediately. Measure absorbance at the end

of 2nd, 3rd, 4th and 5th minute. Then wash the cuvette with distilled water.

6. Estimation of sample activity:

Pipette 20 μl of sample and 1000 μl of „working solution“ ,start up stopwatch, mix

the solution and insert it into the cuvette immediately. Measure absorbance at the end

of 2nd, 3rd, 4th and 5th minute. Then wash the cuvette with distilled water.

7. Enter the obtained values into a table in the computer (LD, summer semester) and

compare the LD value with reference values and evaluate.

Reference value: less than 4.2 μkat/l

51

2) Alanine aminotransferase and aspartate aminotransferase in liver

Principle:

The estimation is based on a different light absorption 2,4-dinitrophenylhydrazine of 2-

oxoglutarate and pyruvate acid that originates directly for reaction of ALT, after spontaneous

decarboxylation of oxalacetate for reaction of AST.

Solutions: substrate for ALT (DL-alanine, 2-oxoglutarate, phosphate buffer pH 7.4)

substarte for AST (L-aspartate, 2-oxoglutarate, phosphate buffer pH 7.4)

solution of 2,4-dinitrophenylhydrazine (DNP-hydrazine in HCl)

sodium hydroxide NaOH (0,4 mol/l)

phosphate buffer pH 7.4

Procedure:

1. Pippete solutions into 4 test tubes according to the table:

Test tubes

1 2 3 4

ALT AST

sample blank sample blank

substrate for ALT 0.50 ml 0.50 ml - -

substrate for AST - - 0.50 ml 0.50 ml

2. Put the test tubes into 37oC water bath for 10 minutes.

3. Preparing of the cell extract:

Crush approximately 1 cm3 of liver tissue in a mortar, add 5 ml of phosphate buffer and continue in homogenisation. All homogenate transfere to a centrifugation tube, centrifugate 5min/2,000 rpm (must do the lab technician). The supernatant is our enzyme solution.

52

4. Add enzyme solution into the sample tubes:

enzyme solution 0.10 ml - 0.10 ml -

5. Put all 4 test tubes into the water bath at 37oC for 30 minutes and then add the

solution of dinitrophenylhydrazine:

dinitrophenylhydrazine 0.50 ml 0.50 ml 0.50 ml 0.50 ml

6. Mix the tubes and let them for 15 minutes at room temperature.

7. Add the solution of sodium hydroxide NaOH into all test tubes:

NaOH 5.00 ml 5.00 ml 5.00 ml 5.00 ml

8. Add enzyme solution into the blank tubes:

enzyme solution - 0.10 ml - 0.10 ml

9. Mix the tubes and wait for 5 minutes. Measure absorbance of test tube No. 1 against

No. 2 (it corresponds to the activity of ALT) and absorbance of test tube No. 3 against

No. 4 (it corresponds to the activity of AST) at 506nm.

10. Evaluate the results.

53

3) Estimation of alkaline phosphatase in blood serum

Principle:

Alkaline phosphatase (ALP) cleaves substrate 4-nitrophenylphosphate to 4-nitrophenol and

inorganic trihydrogenphosphate. Enzyme activity corresponds to the amount of 4-nitrophenol

which is estimated photometrically in alkaline environment.

Solutions: buffer (methylglucosamine and magnesium sulfate MgSO4)

substrate (4-nitrophenylphosphate + glucose)

inhibition solution (EDTA in NaOH)

serum (in a small plastic tube - Eppendorf tube)

Procedure:

1. Pipette solutions into two test tubes according to the table: It is necessary to work very attentively. The serum must be pipetted to the bottom of the tube, it must

not stay on the wall of the tube!

Test tube No. 1

sample

Test tube No. 2

blank

buffer 1.00 ml 1.00 ml

serum 0.02 ml -

2. Mix and put both into a water bath at 37oC for 5 minutes:

3. Add substrate to both tubes:

substrate 0.20 ml 0.20 ml

4. Put tubes into a water bath at 37oC for 10 minutes exactly and then stop the reaction

by adding the inhibition solution:

inhibitor 0.50 ml 0.50 ml

5. Add just to the tube No. 2 (blank) 0.02 ml of serum:

serum - 0.02 ml

Both tubes contain the same solutions. The serum was added to the tube No. 2 (blank) after adding the

inhibition solution. The reaction can run only in the tube No. 1. The more enzyme is presented in the

serum the more substrate is cleaved and the more colored product can be obtained.

54

6. Mix the tubes and measure the absorbance (A) of the sample (tube No. 1) against the

blank (tube No. 2) at 420nm.

Calculation:

Calculation of the catalytic concentration of ALP in an examinated serum.

ALP (kat/l) = A × 10.236

Evaluate the result and compare with referentce values.

Reference value: 0.7 – 2.2 μkat/l (children up to 7.0 μkat/l)

55

Enzymology IV

1) Inhibition of succinate dehydrogenase with malonate

Principle:

Succinate dehydrogenase is an enzyme of citrate cycle. It belongs to the riboflavine-linked

dehydrogenases presented in the inner mitochondrial membrane. It converts succinate to

fumarate under normal conditions. Malonate is known as an competitive inhibitor of this

reaction. In the nature conditions this enzyme transfers electrons through the respiratory chain

to the oxygen. In order to measure this reaction in our experiment we have to block the

respiratory chain, particulary the function of cytochromes with KCN, and we supply an

artificial electron acceptor – potassium hexacyanoferrate (III), K3[Fe(CN)6], which is reduced

to potassium hexacyanoferrate (II), K4[Fe(CN)6]. This reduction can be observed

photometricaly, and it goes with coloure changing, fading of a yellow solution.

In this experiment there is possible to observe the kinetic of enzyme reaction easily even

effects of inhibitors.

Solutions: pH of solutions is conditioned on 7.2

sodium succinate (0.2 mol/l)

potassium cyanide (0,1 mol/l), POISON!

potassium hexacyanoferrate (III) (0.01 mol/l)

sodium malonate (0,01 mol/l)

phosphate buffer pH 7.2 (0.1 mol/l)

a sample of frozen heart for preparing enzyme solution

Procedure:

1. Prepare an ice bath. Put several ice cubes and distilled water into 250 ml beaker.

2. Take a calibrated tube and 5x diluted phosphoric buffer, 2 ml of phosphate buffer + 8 ml

of distilled water. Mix it and place into an ice bath.

3. Preparation of the enzyme solution:

Take a small piece of pork heart, put it into a mortar and cut it with a scalpel into small

pieces. Wash the pieces with approximately 3 ml of diluted phosphate buffer and

homogenize the tissue with a pestle. Transfer the crude homogenate into the

centrifugation tube and spin it down for 5 minutes at 2,000rpm. Meanwhile, clean

the mortar and pestle.

56

During the preparation of the enzyme solution, it is desirable to prepare a „basic solution“

(point 5 in this procedure).

4. After centrifugation pour the supernatant carefuly in the waste. Put the sediment that

must be colourless, into the mortar and resuspend it. (Any colour would interfere with

photometric measurements.) Add approximately 3 ml of diluted phosphate buffer and

glass pieces for better homogenisation (use protective glasses!). Transfer the mixture into

the centrifugation tube and spin it down for 5 minutes at 2,000 rpm. After that collect

the supernatant because it is our enzyme solution. Store it in the ice bath because

mitochondria is very sensitive compages.

5. Preparing of a „basic solution“:

Mix 3 ml of potassium cyanide, KCN, 3 ml of potassium hexacyanoferrate (III), K3[Fe(CN)6], and 10 ml of phosphate buffer (undiluted).

6. Pipette the solutions into test tubes according to the table:

Test tube 1 2 3 blank

„basic solution“ (ml) 2.0 ml 2.0 ml 2.0 ml -

sodium succinate (ml) 1.0 ml 1.0 ml - -

sodium malonate (ml) - 0.5 ml 0.5 ml -

distiled water (ml) 0.5 ml - 1.0 ml 3.5 ml

7. Switch on the photometer, adjust the wavelength 415nm, and prepare 4 photometric

cuvettes, stop watch, and 0.5 ml pipette.

8. Add 0.5 ml of enzyme solution to the blank, mix and pour it into the cuvette and adjust A

= 0.000.

9. Add 0.5 ml of enzyme solution into the test tube No. 1, mix and pour it into the cuvette

immediately and measure absorbance at time 0 and start to measure the time. Note the

value of absorbance.

10. Pipette 0.5 ml of enzyme solution into the test tube No. 2 very quickly, mix and pour it

into another cuvette. Place the cuvette into the photometer and measure the absorbance.

The time of measurement must be 30 seconds after the measurement of the test tube No.

1 accurately.

11. Change the cuvettes in a period of 30 seconds. Each cuvette is measured in a period of 1

minute. Make 20 measurements for each cuvette overall.

12. Process the test tube No. 3 which is chacking in the same way. It contains just an

inhibitor, no substrate. Add 0.5 ml of enzyme solution and measure the absorbance in a

period of 1 minute. There can be observed just slight decrease of the absorbance what is a

systhematical error of the method.

13. Write down all absorbance values in the table into the computer and a graphic processing

will be done.

14. Evaluate the results and make a conclusion.

57

2) Estimation of aldolase activity

Principle:

Aldolase is a glycolytic enzyme catalysing the reversible conversion of fructose-1,6-

bisphosphate to glyceraldehyde phosphate and dihydroxyacetone phosphate. Both products

can be evidenced by their reaction with dinitrophenylhydrazine in alkaline media. Hemolysis

influences results due to washing out of enzyme from RBCs.

Solutions: solution of fructose-1,6-bisphosphate (0.02 mol/l, pH 7.4)

solution of monoiodoacetic acid (0.002 mol/l, with NaOH, pH = 7,4)

solution of hydrazine sulfate (pH = 7.4)

collidine buffer (2,4,6-trimethylpyridine, pH = 7.4)

solution of dinitrophenylhydrazine in 2M HCl

trichloracetic acid

solution of sodium hydroxide, NaOH (3%)

serum

hemolytic serum

Procedure:

1. Pipette solutions into 4 centrifugation tubes according to the table:

Test tube No. 1

sample

Test tube No. 2

blank

Test tube No. 3

sample

Test tube No. 4

blank

collidine buffer 0.2 ml 0.2 ml 0.2 ml 0.2 ml

serum 0.2 ml 0.2 ml - -

hemolytic serum - - 0.2 ml 0.2 ml

hydrazine sulfate 0.05 ml 0.05 ml 0.05 ml 0.05 ml

monoiodoacetic acid 0.05 ml 0.05 ml 0.05 ml 0.05 ml

trichloracetic acid - 0,4 ml - 0,4 ml

2. Mix the tubes and place them into the water bath at 37°C for 5 minutes.

3. Add to all tubes 0.1 ml of the solution of fructose-1,6-bisphosphate, mix and place

into the water bath at 37°C for 20 min. The reaction does not proceed in test tubes No.

2 and 4 due to trichloracetic acid.

58

4. Add 0.4 ml of trichloracetic acid even into remaining test tubes No. 1 and 3. An

arisen precipitate is cleared off by centrifugating at 2,500 rpm for 10 minutes.

5. Prepare 4 new thin-walled test tubes, mark them in the same way as the centrifugation

tubes. Transfer 0.5 ml of clear supernatant to new clean tubes.

6. Add to all test tubes 0.5 ml of 3% solution of NaOH and let the mixtures for 5 min at

room temperature.

7. Add to all test tubes 0.5 ml of dinitrophenylhydrazine and mix properly. Place the

tubes into the water bath at 37°C for 5 minutes and then add 3 ml of 3% NaOH and

let samples for 5 minutes at room temperature.

8. Intesity of coloring is measured, the samples against blanks, i.e. No. 1 against No. 2

and No. 3 against No. 4 in 1 cm cuvettes at 540nm.

Estimation of aldolase activity:

[μkat/l] = A x 0,98

Evaluate your results and compare aldolase aktivity in serum and in hemolytic serum.

Reference value: less then 50 nkat/l

59

3) Estimation of xanthine oxidase activity

Principle:

Xanhtine oxidase is a complex molybdenum containing flavoproteine. It is able to convert

hypoxanthine to xanthine, and xanthine to uric acid. It is present in many organs and also in

milk. Its substrate specificity is not absolute and therfore even non-specific substrates can be

oxidated by this enzyme, if a suitable electron acceptor is present. This acceptor is usually the

molecular oxygen which subsequently forms water. In this reaction also the one-electron

reduction product is often formed, i.e. the superoxide anion O2- which has to be

disproportionated by superoxide dismutase and catalase.

In our experiment formaldehyde as a substrate for the xanthine oxidase and the methylen blue

as the first electron acceptor are used. The final electron acceptor is the atmospheric oxygen.

This experiment can be used as a simple test for milk pasteurisation. Higher temperature

causes enzyme denaturation and adding of formaldehyde cannot raise discolouration of

methylen blue.

Solutions: solution of methylen blue, 0,01%

solution of formaldehyde, 2,0%

fresh milk (non-pasteurised)

Procedure:

1. Pipette solutions into 4 test tubes according to the table:

Test tube No. 1 2 3 4

Fresh milk 5 ml 5 ml 5 ml 5 ml

Boil test tubes No. 3 and No. 4 above a burner carefully. Next continue according to

the table:

Methylen blue 5 drops 5 drops 5 drops 5 drops

Formaldehyde 5 drops - 5 drops -

2. Shake well the tubes and place them into the water bath at 37º C. Look where

discolouration can be observed and explain the reason.

3. Take out the test tube where the blue colour disappeared and shake vigorously until

the blue colour is restored. Then continue the reaction in the water bath.

4. You can repeat this cycle several times until all formaldehyde is exhausted. Then

methylen blue will not loose the colour any more. In such a case few drops of

additional formaldehyde will restore this ability again.