- •Federal Agency of Education tambov state university named after g.R. Derzhavin
- •Biochemistry (part I)
- •Tambov 2012
- •I. Theoretical part The subject Of biological chemistry
- •1. Chemistry of proteins
- •1.1. Methods of exctraction and purification of proteins
- •1.2. Functions of proteins
- •1.3. Amino-acid composition of proteins
- •1.4. The structural organization of proteins
- •1.5. Physical and chemical properties of proteins
- •1.6. Classification of proteins
- •1.6.1. Simple proteins
- •1. Albumins and globulins.
- •2. Protamines and histones.
- •3. Prolamins and glutelins.
- •1.6.2. Conjugative proteins
- •1. Proteins containing non-heme iron.
- •Nucleic acids
- •The structure of nucleic acids
- •Test Questions
- •2. Enzymes
- •2.1. The chemical nature of enzymes
- •2.2. The mechanism of enzymes action
- •2.3. Kinetics of enzymatic reactions
- •2.4. Enzyme properties
- •2.5. Regulation of enzyme activity
- •1. Control of the enzyme amount.
- •2. Control of enzyme activity.
- •2.1. The influence of activators and inhibitors on the enzymes.
- •2.3. Chemical modification of the enzyme.
- •2.4. Allosteric regulation.
- •2.6. Classification and nomenclature of enzymes
- •2.7. Enzymes in medicine
- •Test Questions
- •3. Vitamins
- •3.1. Fat soluble vitamins
- •3.2. Water soluble vitamins
- •Vitamin-like substances
- •Test Questions
- •4. Basic principles of biomembranes’ organisation
- •4.1. Structure and functions of membranes
- •1. Phospholipids (90%) - glycerophospholipids and sphingophospholipids:
- •4.2. Transport of substances across membranes
- •2. Facilitated diffusion
- •Test Questions
- •5. Transmembrane transduction of the hormonal signal
- •Test Questions
- •6. Introduction to metabolism
- •6.1. Stages of catabolism
- •III. A common catabolic pathway.
- •6.2. Bioenergetics
- •6.3. Organization and operation of the respiratory chain
- •6.4. Uncoupling of oxidation from phosphorylation
- •6.5. Generation of free radicals in cells
- •6.6. Reactions of the common catabolic pathway
- •6.6.1. Oxidative decarboxylation of pyruvate
- •6.6.2. Citric acid cycle
- •7. Carbohydrate metabolism
- •7.1. Digestion of carbohydrates
- •7.2. Glycogen metabolism
- •7.3. Glycolysis
- •7.4. The inclusion of fructose and galactose
- •In glycolysis
- •7.5. The shuttle mechanisms
- •7.6. Cori cycle
- •7.7. Alcoholic fermentation
- •C6h12o6 → 2 ch3chohcooh
- •7.8. Pentose phosphate pathway (hexose monophosphate shunt)
- •7.9. Gluconeogenesis
- •Synthesis of glucose from the pyruvic acid.
- •Fructose bisphosphatase
- •Glucose-6-phosphatase
- •7.10. Regulation of carbohydrate metabolism
- •Glucose → glucose-6-phosphate.
- •Pyruvate → oxaloacetate → phosphoenolpyruvate
- •7.11. Violations of carbohydrate metabolism
- •Violation of hydrolysis and absorption of carbohydrates
- •Violations of the intermediate metabolism of carbohydrates
- •Quiz Questions
- •II. Laboratory practice Laboratory work 1. The analysis of amino acids and proteins
- •1. Qualitative analysis of amino acid mixtures by thing layer chromatography method.
- •2. Colour reaction of proteins.
- •2.1. Millon’s reaction.
- •2.2. Ninhydrin’s reaction.
- •3. Reactions of precipitation of proteins.
- •3.1. Precipitation of proteins with salts of heavy metals.
- •3.3. Precipitation of proteins with the help of concentrated mineral acids.
- •3.4. Precipitation of proteins by organic acids.
- •Test questions
- •Laboratory work 2. Conjugated proteins: glycoproteins and phosphoproteins
- •1. Phosphoproteins.
- •1.1. Proof of protein component’s present in the casein hydrolyzate.
- •1.2. Proof of phosphoric acid’s presence in the casein hydrolyzate.
- •1.3. Determination of the isoelectric point of casein.
- •1.2. Reaction with diphenylamine.
- •2. Chromoproteins.
- •2.1. Benzidine test for hemin group of hemoglobin.
- •2.4. Proof of carbohydrate’s presence in the egg albumin.
- •Test questions
- •Laboratory work 3. Conjugated proteins: nucleoproteins and chromoproteins
- •1. Nucleoproteins
- •1.1. Isolation of deoxyribonucleoproteins from the spleen.
- •Test questions
- •Laboratory work 4. Enzymes
- •1. Detection of peroxidase in potatoes.
- •2. Detection of pepsin in the gastric juice.
- •3. Hydrolysis of starch by α-amylase.
- •4. Specificity of amylase and sucrase enzymes action.
- •Test questions
- •Laboratory work 5. Identifying the activity of enzymes
- •1. The influence of activators and inhibitors upon activity of enzymes.
- •1. The influence of activators and inhibitors upon α-amylase.
- •1.2. Inhibitory effect of chloride ions on dehydrogenase complex of potatoes.
- •2. Identifying the activity of α-amylase according to Wolgemut.
- •Test questions
- •Inhibitor calimine
- •Laboratory work 6. Vitamins
- •Vitamin a (vitamin a, retinol)
- •Vitamin d (calciferol)
- •3.1. Reaction with concentrated nitric acid.
- •3.2. Reaction with ferric chloride (III).
- •9.2. Reaction with methylene-blue.
- •Test questions
- •Laboratory work 7. Oxidoreductases. Common catabolic pathway
- •1. Comparison of redox-potentials of riboflavin and methylene blue.
- •2. Identifying of catalase activity according to a.N. Bach and а.I. Oparin.
- •Test questions
- •Laboratory work 8. Carbohydrate metabolism
- •2.1. Trommer’s test with copper hydroxide.
- •2.2. Discovery of fructosuria by Selivanov’s test.
- •2.3. Enzymatic method of semi-quantitative identification of glucose in urine with the help of "GlucoPhan" test strip.
- •Test questions
- •References
1.5. Physical and chemical properties of proteins
Proteins are high-molecular compounds, their relative molecular weight varies from 6000 to 1000000 а.m.u. and more. For the definition of molecular weight of proteins we use sedimentation analysis methods, gel chromatography and gel-electrophoresis.
The most typical physical and chemical properties of proteins are high viscosity of solutions, insignificant diffusion, ability tj swell within wide limits, optical activity, mobility in electric field, low osmotic pressure and high oncotic pressure, ability to absorb UV rays at 280 nanometers (this property is used for quantitative definition of proteins). Protein molecules are not capable to inpour through semipermeable artificial membranes, and also biomembranes of vegetative and animal tissues.
In the isoelectric point (рI) the charge of the protein molecule is equal to zero, and proteins do not move in electric field. There are no repulsive forces between molecules, and protein molecules tend to form aggregates. In the isoelectric point proteins are least soluble and easily drop out into sediment. For the majority of animal tissue proteins рI = 5,5 - 7,0. Hb – 6.8, serum albumin – 4.9.
Protein solution is called isoionic if it does not contain any other ions, except the ionized units of amino acids of protein molecule and the ions formed at their dissociation. Protein isoionic point is a value of рН of protein isoionic solution. Isoionic and isoelectric points coincide only in the case when both are equal to 7.
1.6. Classification of proteins
Depending on chemical composition proteins are divided into 2 groups.
Simple proteins are constructed of the amino acids residues and in hydrolysis give only free amino acids.
Conjugative proteins are two component proteins which consist of simple protein and non-protein component (prosthetic group).
1.6.1. Simple proteins
1. Albumins and globulins.
Albumins are simple proteins; are a part of animal and vegetative tissues; are contained in eggs, blood serum, milk, in seeds of plants. They are soluble in water, salting liquids, acids and alkalis. Approximately 75-80 % of osmotic pressure of proteins of blood serum are albumins; their one more function - transport of fat acids.
Globulins are group of animal and plant proteins widely spread in nature. They are globular proteins. They are soluble in weak solutions of neutral salts, weak acids and alkalis. The globulins presented by a-fraction are contained in blood in a complex with bilirubin and with lipoproteins of high density. The fraction of β-globulins includes a prothrombin which is the precursor of thrombin - the protein participating in blood coagulation. The fraction of g-globulins is the most heterogenous. The major g-globulins are immunoglobulins.
The ratio albumin/globulin has a diagnostic importance: in norm it is close to 2, and during inflammatory diseases it is decreases.
2. Protamines and histones.
Protamines are low molecular weight proteins in nuclei of spermatozoons of fishes and birds. They contain 60-85% arginine and possess the basic properties. They are well dissolve in water, acid and neutral medium and precipitate in alkalis. In nuclei of cells they associate with DNA.
Histones are small proteins of the basic character. Their composition has lysine and arginine which amount does not exceed 20-30%. There are five various types of histones: H1, H2A, H2B, H3 and H4. These proteins are mainly in nuclei of cells. They take part in the structural organization of chromatin.