Biochem -102, 3(2-1)

Elementary Biochemistry

Theory A general introduction to the science of biochemistry. Ionization of water,

weak acid and weak bases, pH, buffers, diffusion, osmosis and osmatic pressure. Enzymes: Classification, nomenclature, characteristics, coenzymes, cofactors and prosthetic groups. Mechanism of enzyme action. Enzyme inhibition. Carbohydrates: Classification, characteristics, aerobic and anaerobic oxidation of glucose, biological functions of carbohydrates. Lipids: Composition and classification, structures of saturated and unsaturated fatty acids and their properties, characteristics of fats and oils, general metabolism of fats and oils. Proteins: Composition and classification, characteristics and classification of amino acids, peptides and levels of structural organization of proteins, physiological function and general metabolism of proteins. Nucleic acids: Chemical composition, structures of DNA and RNA. Functions of DNA and different types of RNA in the cell.

Introduction to Biochemistry

Chemical Structures, reactions, principles and machanism behind such interactions with all aspects around Chemistry Biomolecule’s-Chemical Structures, reactions (Metabolism), principles and machanism behind such interactions with life in all its deverse forms and aspects either directly or indirectly BioChemistry Molecular Level “Molecular logic of Life in all its diverse forms can be explained by Biochemistry”

•Deals with metabolic processes in living tissues a material

called Protoplasm (basis of all life)

•These reactions in Normal way HEALTHY But

Disorganization SICKNESS/ DEATH

•All components that make life are themselves inanimate but

combinations makes life possible.

•Young emerging science in the 20th century but now a major

Discipline dependent on the discoveries of braches of

Chemistry (organic, inorganic, physical , analytical etc), Physiology

Scope and importance of Biochemistry

Now it answers to explanations for the machanisms behind Physiology Medical sciences Physiological, Pharmacolgy, Bacteriology, Pathology

Solutions to clinical problems, remedies to deficiencies like Rickets,

pellegra (B3), Beri-Beri, Scruvy, Aneamia Diagnosis and therapy

Purifying vitamins, hormones, Anti-toxins, vaccines Enzyme inhibitors, Recombinant DNA technology, genetic engineering , cloning , profiling Mysteries in agriculture, industry, research and all life sciences

Books recommended

Principles of biochemistry by Lehninger (4th edition and onward) http://www.irb.hr/users/precali/Znanost.o.Moru/Biokemija/Literatura/Lehninger%20Principles%20of%20Biochemistry,%20Fourth%20Edition%20-%20David%20L.%20Nelson,%20Michael%20M.%20Cox.pdf

Medical biochemistry by Mushtaq Ahmed vol-1 edition after 2008 Cell and molecular biology by Gerald Karp 3rd edition and onward any http://www.btsdl.cc/cell-and-molecular-biology-by-gerald-karp-6th-edition-tf2432083.html

THE CELL CELL THEORY;

1. All living organisms are made up of cells and cellular components. May be

uni/multi cellulars.

2. Basic structural and functional unit of Life

3. All cells are produced from preexisting cells.

Properties ; •A high degree of chemical complexity and microscopic organization.

•Systems for extracting, transforming, and using energy from the environment to do work

•Defined functions for each of an organism‘s components and regulated interactions

•Mechanisms for sensing and responding to alterations in their surroundings.

•A capacity for precise self-replication and self-assembly.

•A capacity to change over time by gradual evolution.

Plasma membranes and cytoplasm “The outer periphery of the cell that separates its internal contents from the surroundings.”

1. Lipid and protein molecules

2. Thin, tough, pliable, hydrophobic barrier

3. Selectively permeable by Transport proteins

4. Signals by receptor proteins

5. Membrane enzymes participate in reactions

6. Flexible in shape and functions less strong bondings

7. Can grow as cells multiply

Cytoplasm: “The internal volume enclosed by the plasma membrane”

1. Aqueous portion cytosol and particular portions gel

2. Rich in enzymes, RNA, metabolites, macromolecules, coenzymes , Ribosomes and

Proteasomes

Movement of materials across membranes

• Cells surrounded by plasma membraneall communications through it

• Dual function of membrane 1) must retain the contents avoid leakage 2) exchange of necessary materials

• Lipid bilayer is best to protect loss of ions, polar, amino acids, sugars, hormones etc

• Movements are passively (gradients, no energy ) and actively (energy needed)maintains net flux (one may exceed other)

Passive transport: Simple diffusion, diffusion through aqueous protein-linked channel,

facilitated diffusion • Spontaneous process in which substances move

from a region of higher concentration to low concentration till equilibrium

• Exergonic energy from random thermal motion / collisions

• If substance Electrolyte movement by chemical gradient or by Electric Potential gradient i.e. by concentrations or by difference between charges electrochemical gradients

• E.g. K+ ions across membrane ++++++++++++++++++++++ 3Na - - - - - - - - - - - - - - - - - - 2K/Cl-

Channels Move ions nerve impulses,

secretions, muscles contractions, cell volume, open stomata etc

• Integral membrane proteins Downhill • Always bidirectional till net flux • Sequence similarities shows this protein has common ancestry • Keeps open/close conformations 1. Voltage gated channels: Conformational changes depends on

difference of ionic charges on 2 sides. E.g. K+ channels 2. Ligand-gated: Conformational changes depends on binding

of a specific (ligand) molecule which is not solute itself. E.g. acetylcholine binds to outer surface to cation channels, cAMP binds inner to Ca++ channel.

C. Elegans (1000 cells), 90 different genesK+ channels

Non-electrolyte diffuses passively…

• Substance must be in high conc at one of membrane

• Membrane Permeable to it Solute must cross aqueous pore without contact with lipid

Solute must cross lipid layer by dissolving

It must have polarity match (NON-POLAR)

Partition coefficient: Ratio of solubility of solute in non-polar solvent (octanol/veg.oil) to that in water, when both solvents are mixed together

2 molecules same P.C, then small uncharged will penetrate faster. E.g. more CO2,O2,H2O, NO etc & Less sugars, amino acids, P-compounds require mechanisms

P.C

Pe

ntr

atio

n c

m/s

ec

sugars

caffein

Small values less permeability

Greater lipid solubility greater penetration

Diffusion of water through membrane best movement example

• Water usually moves rapidly as compared to other ions / polar solutes selectively permeable membrane called OSMOSIS

“Water moves readily across a semi-permeable from a region of lower solute concentration to a region of higher solute concentration”

Demonstrated by placing a cell in a non-permeable solute solution of different concentrations across plasma membranes

Hypertonic /hyperosmotic solution

Cells shrink and own H2O comes out cured by gain of ions

Hypotonic /hyposmotic soltn

Cells swell by gaining H2O from outside cured by loss of ions

Isotonic0.85% NaCl saline

No net flux Temporary but best example of movement and shape changes

Uses

• Digestive tract secrete Liters of of fluid but reabsorbed by osmosis

Animals remain in isoosmotic , but in diahhrea occurs if fails to reabsorb

• Plant cells hypertoniclet H2O inside Turgid (turgor pressure pushes against cell walls) in hypertonic solutions plasmolysis

• Aqua porins in some cells more permeable to water kidney/plant root protein-channels allows this

In congenital nephrogenic diabetes insipidus mutations in aquaporins vassopressins fail no reabsorbtion higher urine excretions

Facilitative diffusions: (fast diffusions by

selectively binding membrane spanning proteins that helps in diffusion)

Solute binds selectively at one end induces Conformational changes exposing it to other end. This transporter binds at a side with solute at one time then to the other side Similar to enzyme-catalysed reactions; Specific to even D/L isomers Obey saturation kinetics i.e. blocked at saturation Regulated Conformational changes are induced (induce fit model) Slow rate 100s to 1000s /sec For Example: GLUT1-5 isoforms, insulin responsive cells in muscles and adipocytes. High glucose insulin GLUT4 more translocated uptake into cells

Active transport like facilitative diffusion transporter proteins Pumps moves

selectively solutes against gradient

V-type pump (only energy changes) & P-type pump (the pumping cycle & conformational changes depends on phosphorylation ) Na-K-ATPase pump

Transportation SYMPORT or Uniports e.g. Na+-glucose co-transporter in kidney & ANTIPORT (species move oppositely) e.g. Na+/H+ exchanger maintains pH

needs energy endergonic + exergonic (metabolic i.e. ATP hydrolysis, light Absorbance, electrons transfer etc). E.g. Retina cells light photons protons rhodopsin sensation

Examples ; H+ pumps = membrane of plants cytosol pH growth control acidification Ca+2-ATPase pump = outside to in in E.R H+/K+-ATPases pump= in stomach pump in H+ and out K+ in exchange

Na-K-ATPase pump tetramers subunits transport + maturation & assembly

2/3rd nerve cells energy and 1/3rd animal cells energy & its electrogenic (separate charges)2K+ in & 3Na+ out

Properties and functions of water

Polar Molecule of H2O

70% by weight Liquid state due to H-bonds

Between others

Between each other

Like dissolves like , so polar solutes and solvents dissolves by interrupting H20-H2O interactions

Higher melting, boiling points and heat of vaporization, great internal cohesion are all due to H-bonds .

Hydrophilics those that dissolve easily Hydrophobics

AS SOLVENT

Ionization of water, weak acids and bases

Proton hoping

Water is weakly ionizable i.e. few molecules

dissociate although its neutral . Many

properties can also be explained by this.

No free proton exists in water but forms hydronium

Ionization Electrical Conductivity and proton hoping makes it fast (cathode/anode movements)

To express ionization quantitatively

For a reversible reaction we know;

Equilibrium constant Keq = fixed and characteristic for any given chemical reaction at a specified temperature. It defines the composition of the final equilibrium mixture

For water;

At 25oC [H2O]=55.5M, for 1L water its 1000/18=55.5

Ionic product of water

E.C=1 x 10-16 M = Keq

At neutral pH we have same concentrations of both H+ & OH-

Thus when H+ is high OH- should be Low. Vice versa

Similarly we also know that;

[H+] [OH-] = 1 x 10-14 M2

Taking –log on Both sides ;

–log[H+] –log [OH-] = –log [1 x 10-14 M2]

Since we know –log [H+]= pH

Thus

pH + pOH = 14 At neutral pH, [H+]= 1 x 10-7 M

pH, pOH, pKa

• pH= - log [H+] where [] represents molar concentration • Strength of H+ in a solution that indicates the measure

of acidic/basic character. Since addition of acids and bases changes ions concentration in indexes or powers of 10 and in decimals. Logarithm changes it into an expressible whole number forms. i.e. 1 x 10-7=[H+]= pH=7.0=neutrality

• pOH= -log [OH-] • pKa = - log Ka (defined as that value of pH at which

the amount of weak acid and its conjugate base are equal) Its helpful in determining the strength of a buffer as pKa±1=buffer capacity, Ka is the dissociation constant of an acid. More values stronger acids highly ionizable.

• The pH of an aqueous solution can be approximately measured with various indicator dyes, including litmus, phenolphthalein, and phenol red, which undergo color changes as a proton dissociates from the dye molecule. Accurate determinations of pH in the chemical or glass electrode method.

• pH is important in clinical sciences acidosis/alkalosis.

•

Buffers

“Are solutions or systems that tends to maintain their own pH when a small amount of acid or base is added to them”

Chemically two types; Acidic = weak acids + conjugate base/salt For Example, CH3COOH/CH3COO- or CH3COONa acetate

buffer or sodium acetate buffer • Basic= weak base or its salts E.G., NH4OH / NH4Cl Hemoglobin is also a buffering system.

• Buffers serve as first line of defence against any foreign invader, enzyme reactions, cell biology, storage, switch on off by maintaining structure-function relationships, microbiology etc.

• Buffer strength is determined by the Molar concentrations of the components making it (0.5 M + 0.5 M= 1.0 M)

• pKa= 4.76 , pH=4.8 (max capacity around) of acetate buffer.

• Among both components weak acids play a significant role in determining capacity and properties of a Buffer.

• pKa ±1 = Buffer Capacity

Buffers…….

• E.g #2: Sodium Phosphate buffers NaH2PO4/ Na2HPO4/ Na3PO4

• CH3COOH + NaOH CH3COONa + H2O • CH3COONa + HCl CH3COOH + NaCl • Common ion effect suppressed the dissociation

of acid base which change pH, neutral salt / water, weak acids as products causing little changes in pH.

• Titration with 0.1M CH3COOH 10mL with 0.1M NaOH to understand buffering action of weak acid

Buffers……. Machanism

Preparation of Buffers

• Prepare a PO4 buffer of pH=7.0 of 250 mL volume with 0.1 M concentration?

Information is required about Potassium and Sodium phosphate salts or acid that has pKa values closest to the required pH.

Information of molar ratios of weak acid and conjugate is required , from handerson-hasselbalch equation.

Molar masses required

Selection ;

• H3PO4=pKa=2.1

• NaH2PO4=pKa=6.8

• Na2HPO4=pKa=12.2

pH = pKa + log [A-]/[HA] 7.0 = 6.8 + log [A-]/[HA] 0.2 = log [A-]/[HA] 0.2/1 = log [A-]/[HA] Taking anti-log on both sides to eliminate log

Anti-log (0.2) = [A-]/[HA] 1.585 / 1 = [A-]/[HA] Thus molar ratio of salt (Na2HPO4) is 1.585 and acid is (NaH2PO4) = 1 Total ratios = 1.585 + 1 = 2.585 Volumes required; Vol of Na2HPO4 = 1.585 / 2.585 x 250 mL = 153.3 mL Vol of NaH2PO4 = 1 / 2.585 x 250 mL = 96.7 mL Check ; 153.3 mL + 96.7 mL = 250 mL

To get information of mass or weigh of the salt and acids for making buffer; We need molar mass of Na2HPO4 . 2 H2O = 178 g NaH2PO4 . 2 H2O = 156 g Amount of Na2HPO4 . 2 H2O = 178 g x 153 mL x 0.1 M 1000 = 2.72 g in total 250ml of salt in buffer solution (dH2O) Amount of NaH2PO4 . 2 H2O = 156 g x 96.7 mL x 0.1 M 1000 = 1.508 g in total 250ml of acid in buffer solution (dH2O) Thus first take 200 mL of dH2O and dissolve Na2HPO4 . 2 H2O = 2.72 g and NaH2PO4 . 2 H2O = 1.508 g and make volume upto 250 mL Check pH Check % error

Problem # 2. Prepare a buffer solution of Na-

Acetate of pH=5.76 , 0.1M of 1 litre volume. (pKa=4.76 of CH3COOH)

Solution

pH = pKa + log [A-]/[HA] 5.76 = 4.76 + log [A-]/[HA] 1 = log [A-]/[HA] Taking anti-log on both sides to eliminate log 10/1 = [A-]/[HA] Total molar ratios = 10 + 1 = 11 Volume of acid = 1/11 x 1000 mL = 90.9 mL Volume of salt = 10/11 x 1000 mL = 909.1 mL

Selection= CH3COOH/ CH3COONa CH3COOH specific gravity 1.052 g/mL, 99%

Amount of CH3COOH = 60 g x 90.9 x 0.1 M 1000 = 0.545 g in total 1000ml of salt in buffer solution (dH2O) Amount of CH3COONa= 82 g x 909.1 x 0.1 M 1000 = 7.5 g in total 1000 ml of acid in buffer solution (dH2O) NOTE= FOR SOLID SALT CH3COONa WE CAN WEIGH BY WEIGHING BALANCE BUT FOR LIQUIDS LIKE CH3COOH WE NEED TO USE VOLUMES EQUIVALENT TO MASS CALCULATED (Info about purity and sp.gravity will help here)

We know CH3COOH specific gravity= 1.052 g/mL, 99%

that is 1.052 g = 1 mL

1 g = 1/1.052 5.45g = 1/1.052 x 5.45

= 5.18 mL

so 99% pure acid required in vol= 5.18 mL 1% = 5.18/99

100% = 5.18/99 x 100 = 5.3 mL of 99% pure is

required to make a buffer

check pH , calculate % Error, check individually while adding components which component plays a vital role in pH.

Numerical assignment (hand written)

• Calculate the concentration of H+ ions in a solution of pH=0.5 ?

• Calculate the OH- ions concentration in a given solution of pH 1.1?

• Calculate the number of molecules of glucose present in 360 grams? • Calculate the number molecules of H2SO4 in 1 mL if its of 1.84 specific gravity?

• What will be the molar volumes of NaH2PO4 and Na2HPO4 required to make a

buffer of 0.5M , if their amounts are 1.25 g and 1 g respectively used for preparing buffer.

• Calculate the amounts of acetic acid and sodium acetate, to prepare pH=7 buffer, of 0.25M and 175mL volume?pKa of acetic acid= 4.76, 96% purity of acid.

• What will be the pH of a solution containing OH- ions 1.34 x 10-4 ?

• What will be the Ka of an acid with pKa value close to 4.8?

What are enzymes ? Biological catalyst Enzymos some catalytic agent derived from Yeast

Catalyst is an agent that accelerates rate of the reaction Vo by lowering the Ea (energy of activation) without itself appearing in the products.

Ea the energy in Cals/mol required to be supplied to the reaction to initiate.

Both Vo and Ea are inversely proportional to each other.

Moles of product formed per unit time is rate of the reaction

Specificity Absolute (1 En 1 S )and relative (1 En (range of substrates) (gluco/hexokinases)

Mode of actions lock and key mechanism & induced fit model

Factors affecting enzyme’s activity or Vo

1. Enzyme concentration: [E] α rate of reaction Vo

• 1st order reaction with increase of one reacting species or factor there is proportional increase or decrease in rate of the reaction

• Depending on hormones, metabolites, other factors increase or decrease [E] i.e. rate of synthesis and degradation (in hours/days)

Factors

2. Substrate concentration:

those following Machelis-menton kinetics start [S] increases the Vo proportionally 1st order reaction when substrate is less and before Km value.

Effect of temperature: within limited range i.e. due to denaturation of proteins of enzymes, temperature increase increases Vo (Q10 principle, 30oC valid)

• Since temperature α K.E α Vo collisions of reactants • But maximum activity seen at each enzymes’ optimum temperature. • Plants tolerate max at 60oC and humans 37oC

Effects of pH: within limited range of pH since pH changes the ionic states of proteins

which keep them intact so certain ionic state of enzyme protein structure + function (active site)

• But maximum activity seen at each enzyme’s optimum pH. • Trypsin = 8-9 pH, salivary amylase= 6.4-6.9

Effects of products: A+B C+D (if reversible) • Reaction may proceed more faster if products removed or reactant increased • Reaction rate may slow if products has similarities with substrate

Cofactors and inhibitors: cofactors (bridge S active site) and coenymes are required to

make enzyme complete and to increase rate of the reaction e.g. Fe++(cytochrome oxidase, catalase etc), Cu++(cytochrome oxidase), Zn++ (alc. Dehydrogenase, carbonic anhydrase), Mg++(hexokinase, pyruvate kinases etc), Mn++ (arginase), K+ (pyruvate kinases), Ni+ (urease)

• Organic or metalloorganic (group transferring) NAD, FAD, FMN, CoASH, etc coenzymes

• Apoenzyme (protein part) cofactors (tightly bound, prosthetic groups) holoenzymes • Inhibitors interfere

CLASSIFICATION OF ENZYMES

OTHLIL

Classification

HEXOKINASES = 2.7.1.1 (ATP; D-Glucose-6-Phosphate Transferases

“Ase” after substrate Lipase, urease, proteases

Machanism = transmethylase, oxidases etc

Trivial names= No relationship but may be latin etc. Pepsin, trypsin, chymotrypsin

IUBMB

4-digits + systemic name + units

i.u.= 1 umole [S][P]/min at 30oC at opt pH. Katal = mole of substrate without 30oC

More than 2 names = succinyl-coA synthatase OR succinate thiokinases

Types of inhibitions of Enzymes

Reversible inhibitions

IRReversible inhibitions

IAAThiols DIPF Acetylcholine esterases Heavy metals (Ag+) EnZ-SH (Mercaptides) Ampicillin transpeptidases (C.W)

Competitive enzyme inhibitions

When resemblance in structure between the [I] and [S] •Binds to some or all free enzymes •Km and Vmax changes •Lowers the [ES] complex •Can be reversed by increasing [S] Important as metabolic antagonists, chemotherapy against bacterial, viral and malignant cancer cells For example; Succinate dehydrogenase

tRNA (puromycin), glutamine(azaserine), Met (ethionine)

In cell wall synthesis and DNA synthesis and stops cells from dividing

•No resemblance with substrate

•Inhibitor doesn’t binds the active site but

sites other than active site.

•Binds to both [E] and [ES]

•Causes change in the 3-D structure of

active sites

•No effect of increasing [S]

For examples ;

1. Ions of heavy metals

2. EDTA chelates Ca++

3. Fluoride removes Mg++

Binds to only [ES] complex.

Makes [ESI] complexes

Not reversed by adding excess [S]

but may be by changes like pH,

temperature, etc

For example; Where two or more

substrates are required like DNA

polymerases being inhibited by

ddNTPs

Isozymes / iso-enzymes

Enzymes that catalyze same reaction (same organism) but are Physically & Chemically distinct as produced at different locations.

Lactate dehydrogenase (LDH I-V) Creatine Kinase (CK 1-3)

Each 34,000 Mol. Wt on electrophoresis

Chemically distinct

LDH

2 genes seperately encoding 2 polypeptides (H & M types)

Combines into a 4-polypeptides unit

LDH-1= H4 (heart), LDH-5=M4 (skeletal muscles)

CK/CPK

2 polypeptides B & M

Combines into Dimer

CK-1= BB (Brain), CK-2=MB (Heart myocardium), CK-3= MM

LDH-2 & CK-2= Myocardial Infarction in serum appears (DIAGNOSIS)

Lungs, kidney, heart ,liver etc

Brain, gonads, heart ,retina, muscles etc

Ribozymes

“Certain RNA molecules have catalytic properties similar to Enzyme therefore called ribozymes”

Substrates Others RNAs or part of ribozymes itself

Therefore believed to be first gene first enzyme evolutionary. As in HIV viruses reverse transcriptasescDNA

Examples of ribozymes

1.414 bp RNA in venomes of tetra himena catalyses self elongation from host nucleotides

2.M1-RNA in Rnase-Pprecursor t-RNA cleavge 3.RNA in self splicing of spliceosomemRNA 4.Small RNA viruses of plants 5.RNA part of rRNA in Ribosome

peptidyltransferase activity

Iron-protoporphyrin-rings heme Catalase H2O2H2O + O2 Benzidine test (Blood detection)

Proteins and Amino

acids

Proteins are complex organic nitrogenous compounds made up of building blocks called amino acids linked by a covalent linkage called peptide bond

>300 amino acids known 20 participate in Protein synthesis standard/primary/normal amino acids (encoded by gene/codons) Proteogenics22Pyrolysine (methanogenics) &

Selenocysteine (25 in Eukaryotes and prokaryotes)

9 Essential amino acids & 11 non-essential

Non-standard/ secondary amino acids

Not in proteins usually but have essential metabolic and physiological roles 1) Pentothenic amino acidVit-B5 Co-enzyme A 2) GABAgamma amino butyric acidNeurotransmitter

α-Carbonα-COOH & α-NH2 19-α- & 1 – Beta amino acid=proline

Uses and types

1. Plastics 2. Drugs 3. Chiral catalysts 4. Food supplements

Aliphatics, acyclic, aromatic (tyrosine=UV absorbance)

Some are non-polar (Hydrophobic clusters)= glycine, methionine, proline etc

Some are polar (uncharged)= serine, asparaginine, etc

Some are polar (charged)= Aspartic acid as –VE , Lysine as +VE

Peptide bond

DIPEPTIDE

If α-COOH of 1st amino acid condenses with the α-NH2 of other adjacent Amino acid Acid-amide bond (Peptide)

• Amino acid residues • Unusual peptide bond

(GSH=γ-glu-cys-gly) • Polypetides (>10 a.a) • Proteins (M.W>10,000)

H-Bonding by (-CO-NH-)

Classification of proteins

1. Simple proteins 2. Conjugated 3. Derived proteins

Based on function

Based on axial ratios

Fibrous (long thread like) proteins e.g. collagen, keratin

Globular (spheroid) proteins e.g. albumins,IG

Based on functions

1.Catalytic proteins 2.Regulatory 3.Structural 4.Transport 5.Immune proteins 6.Contractile 7.Genetic 8.Storage

Enzymes

Hormones (Insulin, GH)

Collagen, Keratin

Transferrin, albumin

Antibodies, IGg

Actin & Myosin

Histones , ssDBP

Casein = Milk Ovalbumin= Egg

3. Derived proteins

Primary derived proteins Secondary derived proteins

When some / all cross linkages that holds the protein molecular structure intact are cleaved

pH,Temperature, ultrasonics, shake vigorously, alcohols etc

Intermediates of progressive hydrolysis of proteins by enzymes/acid

Proteins Proteoses Peptones

Polypeptides oligopeptides

Dipeptide Amino acids

Simple Proteins

Made up amino acids and its derivatives only i.e. these are hydrolysis end products

1. ALBUMINS 2. GLOBULINS

Serum albumin, Ovalbumin, legumelin, lactalbumin etc

Serum Globulin, Ovoglobulin, legumin, lactoglobulin, myosin.

Examples

3. Globins 4. Histones

5. Collagen

Histidine residues not basic Slightly basic pH Heme is Same in all but globins differenthemoglobin

Proline , glycine hydroxy-proline, cysteines Resistant Keratin = shell, hoofs Elastin=arteries,tendon

Rich in basic proteins like arginine and lysine +ve charge nucleic acids –Ve chargenucleosome

Conjugated proteins

Made up amino acids and its derivatives covalently linked to a non protein part called prosthetic group

Glycoproteins

Chromoproteins Metalloproteins

Phosphoproteins

Lipoproteins

Nucleoproteins

Practical

• Determination of pH value of biological fluids. Preparation of buffers of definite pH. Estimation of optical activity by polarimetry. Qualitative analysis of carbohydrates. Qualitative analysis of urine for normal and abnormal constituents - albumin, acetone bodies and sugar. Estimation of glucose in biological fluids. Determination of acid, sponification and iodine values of fats/oils. Estimation of lactose and casein in milk.

Outlines to cover • Preparation of solutions, normal,

molar, ppm, percentage, dilutions,

determination of pH of solution,

buffer preparation,

Concentrations, Molarities, pH, pOH

Determination of protein and oil contents

• Biochemical tests for proteins and amino acids

• Biochemical tests for oils and fats

Estimation of glucose , fructose and starch

(Carbohydrates biochemical tests)

• Molisch test, iodine test, barfoed, benedicts , salivanoff tests , phenyl hydrazine test etc

• Estimation of lactose and casein in milk

Determination of optical activities of sugar solutions

• Saponification and I2 values of fats and oils

Determination of acetone and albumin in body solutions

Basics to start

• Milligrams (mg)= 1000th part of a gram 10-3 g or 0.001 grams

• Kilograms = 103 grams or 1000 g • Parts per million (ppm)= measures smallest levels of

pollutants in air, water, body fluids. i.e. mass of a pollutant and a solution

Ppm= 1,000,000 Mc/Ms (Mc = mass of component, Ms= mass of solution)

E.G. = 1 mg/ Kg as mass per unit of mass

Ppm……

• E.g # 2; mg / Lit as Mass per volume • Parts per billions (ppb)= 10-9

• Parts per trillions (ppt)= 10-12

• Parts per quadrillions (ppq)=10-15

1 L ≡ 1 dm3 ≡ 1000 cm3 1 L ≡ 0.001 m3 ≡ 1000 cm3 1 m3 =1000 L

• Distilled water d H20, dd H20, ddd H20 (Distilled and deionized)

Molarity (M), Normality(N), molality(m)

• Molarity: Number of Moles of solute dissolved in one Litre (1 dm3 =1000cm3) of solution. Unit=moles/ dm3

M=n/L . [Mole = given mass/ molar mass] • Molality: Number of Moles of solute dissolved in

one Kilogram of solution.

• Normality: Number of gram equivalents of solute dissolved in one Liter of solution.

• 1 mole of Glucose = 180.2 g of glucose

• Molar mass / # of replaceable H+ or OH- ions

OR (Gram equivalent weight)

Molar mass / valence number

• 1 gram equivalent of H2SO4 = 98/2 = 49 g= 1 g.eq

So 49 g of H2SO4 dissolved in 1 Liter= 1.0 N H2SO4

Also ; Ca(OH)2= 74/2 = 37 g = 1g.eq/Lit = 1 N solution

• Avogadro's number = 6.022 x 1023 atoms/molecules in a mole

• Bases = Proton Acceptor e.g. a monoacidic base NaOH ↔ Na+ + OH-

• Acids = proton donor e.g. a monobasic acid HCl ↔ H+ + Cl-

• Buffer = solutions or systems that maintain their own pH when a small amount of acid or base is added to them. E. g. NaH2PO4/Na2HPO4 sodium phosphate buffer

Solute , solvent , solution and mixture

• Solute : Segment of a solution that is in lesser amount and determines

the strength of a solution. Its is always dissoolved in other substances. E.g. solid, liquid, gas

• Solvent : Segment of a solution that is in greater amount and

accomodates a solute usually. E. g. Gas or liquids

• Solution: A homogenous mixture of 2 or more substances either solids,

liquids or gases or combinations of both. Some interactions or linkages do develop, hard to see through when soluble E.g. soft drinks, 10 % salt solution

• Mixture: in mixtures components cannot dissolve but are combinations

of each other. Solution is a mixture but mixture cant be a solution. Can be easily separated by physical means like heating , filtration etc. No interactions exist e.g. sand mixed with salts.

Precision and accuracy and specific gravity

• Precision = degree to which an instrument or process repeats the same value or How much reproducible the experiment is.

• Accuracy = degree of closeness to true values

• E.g. refrigrator set at 38 degrees gives

38.1, 38.5 , 38.7, 38, 38.5, 38.9, 39 etc (More accuracy less precise)

• Specific gravity= weight of substance in 1 mL of solvent/solution e.g. 1.84 g/mL H2SO4

Ratio of density of substance/density of refernce substanc

• Absorbance = bulk phenomenon, homogenous or uniform, as a fluid or gas dissolves in a liquid or solid absorbant distributes thoroughly. E.g. ink drop in water glass

• Adsorbance = surface based phenomenon, higher at surfaces not homogenous, at start increases but then become slow at equillibrium , adheres only physically. E.g. gasses trapped in the spaces of stars

%composition, analyte, titration, standard solution, blank, sample,

w/v, w/w, v/v. The target to qualify or measure in a mixture Volumetric method of determining the concentration of an

unknown solution whose volume is known by the help of a standard. Sometimes indicators help. E.g. acid-base titrations, redox-titrations etc

Whose concentration is known Contains everything present in a reaction mixture except

your target analyte, gives zero readings of an experiment. Sample is the mixture containing unknown amounts of

your analyte and its analysis is to be done. It is always a crude.

Properties and functions of water

Polar Molecule of H2O

70% by weight Liquid state due to H-bonds

Between others

Between each other

Like dissolves like , so polar solutes and solvents dissolves by interrupting H20-H2O interactions

Higher melting, boiling points and heat of vaporization, great internal cohesion are all due to H-bonds .

Hydrophilics those that dissolve easily Hydrophobics

AS SOLVENT

Ionization of water, weak acids and bases

Proton hoping

Water is weakly ionizable i.e. few molecules

dissociate although its neutral . Many

properties can also be expalined by this.

No free proton exists in water but forms hydronium

Ionization Electrical Conductivity and proton hoping makes it fast (cathode/anode movements)

To express ionization quantitatively

For a reversible reaction we know;

Equilibrium constant Keq= fixed and characteristic for any given chemical reaction at a specified temperature. It defines the composition of the final equilibrium mixture

For water;

At 25oC [H2O]=55.5M, for 1L water its 1000/18=55.5

Ionic product of water

E.C=1.8 x 10-16 M = Keq

At neutral pH we have same concentrations of both H+ & OH-

Thus when H+ is high OH- should be Low. Vice versa

Similarly we also know that;

[H+] [OH-] = 1 x 10-14 M2

Taking –log on Both sides ;

–log[H+] –log [OH-] = –log [1 x 10-14 M2]

Since we know –log [H+]= pH

Thus

pH + pOH = 14

At neutral pH, [H+]= 1 x 10-7 M

Similarly –log [OH-]= pOH

pH, pOH, pKa

• pH= - log [H+] where [] represents molar conc • Strength of H+ in a solution that indicates the measure

of acidic/basic character. Since addition of acids and bases changes ions concentration in indexes or powers of 10 and in decimals. Logarithm changes it into an expressable whole number forms. i.e. 1 x 10-7=[H+]= pH=7.0=neutrality

• pOH= -log [OH-] • pKa = - log Ka (defined as that value of pH at which

the amount of weak acid and its conjugate base are equal) Its helpful in determining the strength of a buffer as pKa±1=buffer capacity, Ka is the dissociation constant of an acid. More values stronger acids highly ionizable.

• The pH of an aqueous solution can be approximately measured with various indicator dyes, including litmus, phenolphthalein, and phenol red, which undergo color changes as a proton dissociates from the dye molecule. Accurate determinations of pH in the chemical or glass electrode method.

• pH is important in clinical sciences acidosis/alkalosis.

•

Buffers

“Are solutions or systems that tends to maintain their own pH when a small amount of acid or base is added to them”

Chemically two types; Acidic = weak acids + conjugate base/salt For Example, CH3COOH/CH3COO- or CH3COONa

acetate buffer or sod.acetate buffer • Basic= weak base or its salts E.G., NH4OH / NH4Cl Hemoglobin is also a buffering system.

• Buffers serve as first line of defence against any foreign invader, enzyme reactions, cell biology, storage, switch on off by maintaining structure-function relationships, microbiology etc.

• Buffer strength is determined by the Molar concentrations of the components making it (0.5 M + 0.5 M= 1.0 M)

• pKa= 4.76 , pH=4.8 (max capacity around) of acetate buffer.

• Among both components weak acids play a significant role in determining capacity and properties of a Buffer.

• pKa ±1 = Buffer Capacity

Buffers…….

• E.g #2: Sodium Phosphate buffers NaH2PO4/ Na2HPO4/ Na3PO4

• CH3COOH + NaOH CH3COONa + H2O • CH3COONa + HCl CH3COOH + NaCl • Common ion effect suppressed the dissociation

of acid base which change pH, neutral salt / water, weak acids as products causing little changes in pH.

• Titration with 0.1M CH3COOH 10mL with 0.1M NaOH to understand buffering action of weak acid

Buffers……. Machanism

General Lab safety procedures • Students are allowed in the laboratory only in the

presence of a tutor.

• Before entering the laboratory, you need to wear a laboratory coat and soft shoes (closed).

• In the laboratory, you need to work carefully, avoid unnecessary conversations and keep your workplace clean.

• Please read the warning signs and symbols placed on the reagents prepare an assignments.

• Preparations and reagents must not be examined by taste.

• Eating and drinking are not allowed in the laboratory.

• Use distilled water, electricity and gas efficiently.

• Take particular caution when handling concentrated acids, bases, poisons and flammable liquids. Concentrated acids, bases and poisons should only be collected by dipping the pipette. Used acids and bases should be poured out into the sink in such a manner as to avoid burns caused by drops of liquid deflected from the wall of the sink (while pouring, hold the mouth of the vessel as close as possible to the drain, gently rinse with water).

• Flammable liquids should be used on premises without

ignited burners or other sources of open flames. They should be stored in tightly sealed vessels.

• Cover your wounds and cuts. • Label each container and keep records like lab books.

• The gas installation should be used with caution. Unnecessary burners should be immediately turned off. When wash with alcohols always dry before coming close to falmes.

• In the case of burns to the skin, mouth or eyes, immediately wash the corrosive liquid with plenty of tap water and notify the tutor. Then, neutralize the acids with 5% sodium bicarbonate, and the bases with 1% acetic acid. Compounds used for neutralization can be found in each laboratory.

• Avoid woolen wears, long hairs opened, nails cutted.

• Be-aware of using all instruments and chemicals by reading SOPs and MSDS.

• Place hazardous and flammable liquids and bottles at a bottom space below your standing position. Any instance should be informed to the supervisor first.

• Disposals and disposal areas should be defined. Avoid sharp edges.

• Before leaving the lab, the workplace, reagents and equipment should be put in order. Wash the lab glass. Close the gas valves. Turn off the taps.

MSDS (material safety data sheet) of different chemicals like HCl, ethanol

etc

SOPs to follow like pH meter etc

Safety measures for radiation, chemical, biological hazards etc

(a manual is available in USC health and safety dept website)

Preparation of Buffers

• Prepare a PO4 buffer of pH=7.0 of 250 mL volume with 0.1 M concentration?

Information is required about Potassium and Sodium phosphate salts or acid that has pKa values closest to the required pH.

Information of molar ratios of weak acid and conjugate is required , from handerson-hasselbalch equation.

Molar masses required

Selection ;

• H3PO4=pKa=2.1

• NaH2PO4=pKa=6.8

• Na2HPO4=pKa=12.2

pH = pKa + log [A-]/[HA] 7.0 = 6.8 + log [A-]/[HA] 0.2 = log [A-]/[HA] 0.2/1 = log [A-]/[HA] Taking anti-log on both sides to eliminate log

Anti-log (0.2) = [A-]/[HA] 1.585 / 1 = [A-]/[HA] Thus molar ratio of salt (Na2HPO4) is 1.585 and acid is (NaH2PO4) = 1 Total ratios = 1.585 + 1 = 2.585 Volumes required; Vol of Na2HPO4 = 1.585 / 2.585 x 250 mL = 153.3 mL Vol of NaH2PO4 = 1 / 2.585 x 250 mL = 96.7 mL Check ; 153.3 mL + 96.7 mL = 250 mL

To get information of mass or weigh of the salt and acids for making buffer; We need molar mass of Na2HPO4 . 2 H2O = 178 g NaH2PO4 . 2 H2O = 156 g Amount of Na2HPO4 . 2 H2O = 178 g x 153 mL x 0.1 M 1000 = 2.72 g in total 250ml of salt in buffer solution (dH2O) Amount of NaH2PO4 . 2 H2O = 156 g x 96.7 mL x 0.1 M 1000 = 1.508 g in total 250ml of acid in buffer solution (dH2O) Thus first take 200 mL of dH2O and dissolve Na2HPO4 . 2 H2O = 2.72 g and NaH2PO4 . 2 H2O = 1.508 g and make volume upto 250 mL Check pH Check % error

Problem # 2. Prepare a buffer solution of Na-

Acetate of pH=5.76 , 0.1M of 1 litre volume. (pKa=4.76 of CH3COOH)

Solution

pH = pKa + log [A-]/[HA] 5.76 = 4.76 + log [A-]/[HA] 1 = log [A-]/[HA] Taking anti-log on both sides to eliminate log 10/1 = [A-]/[HA] Total molar ratios = 10 + 1 = 11 Volume of acid = 1/11 x 1000 mL = 90.9 mL Volume of salt = 10/11 x 1000 mL = 909.1 mL

Selection= CH3COOH/ CH3COONa CH3COOH specific gravity 1.052 g/mL, 99%

Amount of CH3COOH = 60 g x 90.9 x 0.1 M 1000 = 0.545 g in total 1000ml of acid in buffer solution (dH2O) Amount of CH3COONa= 82 g x 909.1 x 0.1 M 1000 = 7.5 g in total 1000 ml of salt in buffer solution (dH2O) NOTE= FOR SOLID SALT CH3COONa WE CAN WEIGH BY WEIGHING BALANCE BUT FOR LIQUIDS LIKE CH3COOH WE NEED TO USE VOLUMES EQUIVALENT TO MASS CALCULATED (Info about purity and sp.gravity will help here)

We know CH3COOH specific gravity= 1.052 g/mL, 99%

that is 1.052 g = 1 mL

1 g = 1/1.052 5.45g = 1/1.052 x 5.45

= 5.18 mL

so 99% pure acid required in vol= 5.18 mL 1% = 5.18/99

100% = 5.18/99 x 100 = 5.3 mL of 99% pure is

required to make a buffer

check pH , calculate % Error, check individually while adding components which component plays a vital role in pH.

pH metry Use of a pH meter 2 electrodes 1 cm apart calomel electrode (Hg / HgCl2 amalgum) Reference electrode (filled with 0.1 M HCl ) for comparison purpose with the given solution Working; Given solution certain amount of H+ ions as compared to 0.1 M HCl potential difference e.m.f (Electr Motive Force) electrons flow galvanometer reads

1. Check pH of water 2. Add acid into separate beaker of dH2O and then salt of conjugate base into separate

beaker 3. Check both of the beaker’s pH values (wash before and after always) and note which

one is more close to our required pH, this component is the acid which contributes in pH determination more

4. Now add 1 mL 1M NaOH in each of the two separate beakers and check the pH changes of addition of strong base, note it will be the acid component that will resist maximum and is the component which contributes in buffer strength maximum.

5. Now mix both the component’s solutions and note the pH again , it will be closest to our required now when both components are present , check % error if any.

To prepare N/10 250 mL of H2SO4 solution?

Concepts of molarity , normalty, solute , solvent, solution, standard solution , primary and secondary standard solutions are important alongwith titrations and indicators etc

Preparation of a secondary standard solution and then standardization by titration with o.1N Na2CO3 solution alongwith indicator to confirm what exact is the concentrations made.

0.5g/100mL dH2O or 0.1% in alc, Acidic= reddish orange <3.1 Basic= Yellow >4.4 Mutagenic , methyl orange

protocol 1 ml H2SO4 in 100 mL dH2O

Add it in a burrette for titration and detetrmination of its normality

In a flask add 5 mL of 0.1 N Na2CO3 (seperately prepared by molar mass 100mL)

Add a drop of methyl orange in it.

Dropwise add H2SO4 from burrette till end point appears from yellow to pinkinsh orange or reddish

Repeat 3 times

Note volume consumed to titrate and complete reaction

Use N1V1=N2V2

Once normality known calculate volume of dH2O required to dilute to attain desired normality

Calculations

Unknown H2SO4 : N/10 Na2CO3 (Known) N1V1 : N2V2 N1(?) x 3.53 mL(Supposed) = 0.1 N x 5 mL N1= 0.14N (supposed) Unknown volume H2SO4 : N/10 H2SO4 (Known) N3V3 : N4V4 0.14 x (??) V3 = 0.1 N x 250 mL V3= 178.6 mL of H2so4 solution Volume of dH2O = 250- 178.5 = 71.4 mL

Qualitative analysis of carbohydrates in the given solution(2-5%)

• Molisch’s test (group identification test for carbohydrates) • Iodine test (polysaccharides like blue for starch) • Ammonium sulphate test (dextrin in polysaccharides purple) • Barfoed’s test (mono saccharides from di-) • Benedict’s test (reducing sugars like monosaccharides) • Fehling’s test (for water soluble ketones and differ from

aldehydes) • Pholoroglucinol test (pentoses like ribose) • Seliwanoff’s test (aldoses and ketoses) • Phenyl Hydrazine test (reducing sugars form osazones crystals

shape specific for type of sugars maltose give sunflower shape) • Hydrolysis of Sucrose and Starch (acid / enzymatic hydrolysis) • Physical state , solubility , taste also gives etc.

Furfural formation (for colored end products)

• Sugars treated strong acids + heated dehydration and hydrolysis forms specific intermingled structures FURFURALs depending on furfural reacts with specific coloring compounds colored complex

• For example;

C5H10O5 C5H4O2 (Furfural)-3H2O H2SO4

Molisch test (group test)

• For free & conjugated carbohydrates(glycoproteins)

• Reagent contains = 1g alpha naphthol (in EtOH) + H2SO4 (forms furfural and condenses with alpha naphthol)

• Procedure;

3 mL G.S test tube 2-3 drops of molisch reagent mix 3 mL conc H2SO4 from sides rotate but don’t shake presence of hydrolysed reddish violet ring at the junction of 2 solutions carbohydrates present

Iodine test (polysaccharides)

• Reagents = I2 + KI (self oxidizing by atmospheric/inhibitor) + HCl (acids for oxidation) adsorption

• Adsorption reversed by alkali

• Adsorption occurs in larger molecules at least 8-Carbon chain

• Sometimes heating also reverses adsorption

Procedure: 3 ml G.S+ 1 drop conc.HCl+1 drop I2 solution.

Results: starch Blue(amylose), Dextrin reddish purple,

Glycogen (amylopectin like) reddish brown, no color no polysaccharides

Benedict’s test (for reducing / non) semi-quantitative

• Free aldo/keto + some disacharides reduces metals like CU+2

CU+

• Sodium carbonate + sod.citrate (avoids Cu ppt)+ CuSO4

CuSO4 Cu++ + SO4 Cu+2 + HOH CU(OH)2 BLUE CU(OH)2 CU+2 + 2OH-

CU+ + amount of reducing sugar (CH2O) CU2O (red ppt) cupric oxide

Detects glucose in urine in clinical labs

Benedict test

• 5 ml benedicts reagent + 8 drops of G.S + mix + boil 2 min + cool

• Results :

Green 0.1-0.5 %

Yellow 0.5 – 1.0%

Orange 1.0 – 1.5 %

Red 1.5- 2 %

Brick red >2%

Barfoed’s test (differ mono/disach)

• Reagents : Cu-acetate and glacial acetic acid (weak)

• Principle: its also reduction metals like CU+2

CU+ in weak acid presence (acids promote oxidation not reduction)mono being strong reducers gives fast reduction even environment not favourable and di gives slow reduction time dependent

Barfoed’s test

• Procedure :

• 2 ml barfoeds reagent + 2 ml G.S + mix + note time + start boiling in water bath

• If red ppt / reddish orange in 2-5 mins boiling + cool monosaccharide

• If ppt after 5-15mins boil disaccharides

Salivanoff’s test (+ve for ketose)

• Also called resorcinol test (coloring agent in reagent mix for furfural reaction) along with HCl

• Principle: Carbohydrates+ HCl furfural only ketohexose + resorcinol cherry red colored complex +ve for ketohexose (Fructose/ sucrose) glucose will be faint pink

• Procedure: 3 ml salivanof reagent + 1 ml G.S + mix + boil for 30 – 60 secs cherry red / dark pink (ketohexose)

Osazone / Phenyl hyrazine test (specific for many reducing mono-disaccharides)

• Reagents : phenyl hydrazine+ Na-acetate (1:2w/w) • Glucose etc hydrolysed C6H5NH2 +ve result

Glucosazone (bundle grass needle crystals)+ NH3 + H2O

• Procedure: 5 ml G.S + 0.3g osazone mix + 3 drops gl. Acetic acid + boil + cool dry on filter paper observe under microscope

• Results : yellow bundle grass shaped (glu/fruc), galactose (needle shaped/flufy), maltose (petals sunflower), lactose (puff ball)