- •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
I. Theoretical part The subject Of biological chemistry
Biochemistry is a science about chemical bases of processes of vital activity, studying chemical components of living cells, and also reactions and processes in which they participate. Its main task is the establishment of communication between a molecular structure and biological function of chemical components of living organisms.
The subject of medical biochemistry is the chemical processes occurring in a human body in norm and pathology, diagnostics and forecast on the basis of biochemical researches.
1. Chemistry of proteins
Proteins are high-molecular nitrogen-containing organic compounds, which molecules are constructed of amino acids residues. Amino acid units linked together with peptide bonds.
Proteins compound the basis of both frames and functions of live organisms. Natural proteins are constructed of 20 various amino acids. These amino acids can be combined in the most different sequences, therefore they can form about millions various proteins. They provide existence of about thousand different living organisms, ranging from viruses to humans. Each organism is characterized by a unique number of proteins.
The content of proteins in various tissues of one organism is unequal. So, in a human body there are proteins in % from dry weight: in muscles - 80, in brain - 45, in bones - 20.
Element composition of proteins in terms of dry weight: C - 50-54 %; Н - 6,5-7,3 %; O - 21-23 %; N - 15-17 %; S - up to 0,5 %.
As a part some proteins contain in small amounts phosphorus, iron, manganese, magnesium, iodine, etc.
The amount of nitrogen is rather constant in all proteins (about 16 %), therefore it is possible to define the quantity of protein in biological objects by the protein nitrogen.
1.1. Methods of exctraction and purification of proteins
Some physical and chemical agents denature proteins. Denaturation is the loss of the secondary, tertiary and quaternary structure of a protein. This leads to a loss of some properties - solubility, biological activity.
Therefore special "sparing" methods are developed for the isolation of proteins.
Process begins with homogenization of biological material. This is grinding to the destruction of cells and subcellular structures. For this purpose we use pistillate or knife homogenizers, ball grinders, ultrasonic sound, method of tissue alternate freezing and thawing, method of "nitrogen bomb".
Then we make extraction of proteins by buffer mixtures with certain values of рН, organic solvents. The majority of proteins well dissolve in dilute solutions of salts.
For a fractionating and purification of proteins we use the following methods.
Salting-out is sedimentation of proteins from solution by adding solutions of salts alkaline and alkaline-earth metals. This method is used in clinical practice in the analysis of proteins of blood serum, for example, for division of globulins (drop out in a deposit at 50 % saturation of solution of ammonium sulphate) and albumins (in 100 % saturation).
Electrophoresis is a movement of charged dispersed particles in an electric field. Direction and speed depend on the charge and size of molecules. It is applied in clinical medicine in the analysis of proteins and peptide mixtures, blood serum.
Ultracentrifugation is a method of division of liquid disperses mediums to components under the influence of centrifugal force.
Chromatography (from Gr. chroma - colour) - a physical and chemical method of division and analysis/ Chromatography based on the distribution of mixture components between two phases - motionless (sorbent) and mobile (eluent).
We distinguish the following types of chromatography:
Adsorptive chromatography. Division of formulation constituents is based on their different sorbtion on solid adsorbent.
Distributive chromatography. The solid phase serves as support for a stationary fluid phase. Paper chromatography is a subtype of it.
Ion-exchange chromatography. We use suitable ion-exchange resin with the functional groups of which part of proteins interchange and stay on column while other proteins are unconstrained and eluate from it.
Gel chromatography, or a method of molecular sieves. It is based on the ability of small molecules to inpour into gel pores whereas the big molecules remain outside, moving together with a mobile phase downwards along a column. It allows dividing proteins with different molecular weight.
Perspective kinds of chromatography are High Performance Liquid Chromatography (HPLC) and gas chromatography. The chromatography is one of the basic methods of biochemical researches. In clinical laboratories it applies to division and analysis of amino acids, proteins, carbohydrates, phospholipids, steroids in blood plasma, tissue extracts, urine.