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Table of contents
Module 2. The common mechanisms of metabolism. Metabolism of carbohydrates, lipids and proteins. Regulation.
Substantial module 2. 1. The role of enzymes and vitamins in metabolism"
Topic 2.1. The control of original knowledge level. Subject and task of biochemistry.
The investigation of protein structure and physical-chemical properties.
Quantitative definition of protein by the biuretic method. The proof of protein
nature of enzymes. ……...... P.3-6
Topic 2.2. The investigation of enzymes structure, physical-chemical properties and
conditions of action ………………………………………………………….. P 7-9.
Topic2.3. Determination of enzyme activity. Investigation of enzyme catalysis kinetics …..P.10-12
and activators and inhibitors influence on enzyme activity.
Topic 2.4. Investigation of the vitamins coenzyme form role in catalytic enzymes activity. P.13-15
Topic 2.5. Investigation of the vitamins and vitamins coenzyme form function ……………...P.16-18
In different biochemical processes.
Substantial module 2. 2“ Basic concepts of metabolism, bioenergetics.
Topic 2.6. Investigation of oxidative phosphorylation and ATP synthesis. Inhibitors ………P.19-22
and uncouples of oxidative phosphorylation.
Topic 2.7. Bioenergetics and general pathways of a metabolism. The investigation of…….. P.23-25
citric acid cycle action.
Substantial module 2. 3 “Metabolism of carbohydrates, its regulation”.
Topic 2.8. Investigation of carbohydrate digestion peculiarities. Glycogen biosynthesis P. 26-29
and degradation. Conversion of monosacharides to glucose in the liver.
Topic 2.9. Anaerobic oxidation of carbohydrates. Glycolysis. Synthesis of glucose –
gluconeogenesis. ……………………………………………………………… P. 30-32
Topic 2. 10 Investigation of aerobic oxidation of glucose. Pentose phosphate pathway….. P. 33-35
of glucose conversion.
Substantial module module 2. 4“Metabolism of lipids, its regulation”.
Topic 2. 11 Structure and functions of cellular membranses. P.36-40
Topic 2. 12 Investigation of lipids digestion peculiarities. Possible disturbanses of P. 41-44
exogenic lipids digestion, absorbtion and transtort.
Topic 2. 13 Investigation of fatty acids and keton bodies metabolism. β-oxidation of fatty P. 45-49
acids. Cholesterol synthesis and steroid metabolism. Disturbances of lipid
metabolism atherosclerosis. Topic 2. 14 Investigation of fatty acids, triacylglycerol and phospholipid synthesis. ….. P. 50-52 Disturbances of lipid metabolism: obesity; lipid dystrophy of the liver.
Substantial module 2. 5 “Metabolism of amino acids, its regulation”.
Topic 2.15 Investigation of gastric juice chemical composition. Studies of proteins P. 53-57
digestion peculiarities.
Topic 2. 16. Studies of amino acids transformation (deamination, transamination…………. P.58-65
decarboxylation) Investigation of separate amino acid metabolism pathways.
synthesis. Disturbances of amino acid metabolism
Topic 2. 17 Investigation of ammonia detoxication and urea synthesis mechanisms. P. 66-68
. .
Questions for Module 2 P.69-71
Task for control module preparing. P.72-75
Topic 2.1. THE METHODICAL GUIDELINES FOR PRACTICE ACTIVITY ON THE THEME:
The control of initial knowledge level. Subject and task of biochemistry. The investigation of protein structure and physical-chemical properties. Quantitative definition of protein by a biuretic method. The proof of protein nature of enzymes.
Biomedical importance:
Thousands of proteins present in the human body perform functions too numerous to list. These include serving as carriers of vitamins, oxygen, and carbon dioxide plus structural, kinetic, catalytic, and signaling roles. It thus is not surprising that dire consequences can arise from mutations either in genes that encode proteins or in regions of DNA that control gene expression. Consequences equally adverse can also result in deficiency of cofactors essential for maturation of a protein. Ehlers-Danlos syndrome illustrates a genetic defect in protein maturation and scurvy a deficiency of a cofactor essential for protein maturation Gross changes in the secondary-tertiary structure of proteins that occur independently of changes in primary structure also are responsible form major diseases. Diseases characterized by significant alterations in secondary-tertiary structure include the prion diseases Creutzfeldt-Jakob disease, scrapie, and bovine spongiform encephalopathy ("mad cow" disease), each characterized by pathologic neurological changes that result from deposition of insoluble proteins in amyloid fibrils composed of continuously hydrogen-bonded β-sheets.
Proteins play the central role during living of cells and forming of the cellular structures. The analysis of the contents in blood of the certain proteins and enzymes is widely used in the diagnostic purposes. At diseases of a liver diagnostic inspection by all means includes electrophoretic definition of the relative contents of albumins and globulins in the plasma of blood. The analysis of the contents lipoproteins and antibodies is usually used at diagnostics of specific types hypoproteinemia and immune infringements. Detection even small amounts of fiber serve in urine the important parameter of disease of kidneys.Now familiarize with the purpose of occupation, think over them, acquire necessity of their studying.
The purpose:
To develop skills in qualitative and quantitative determination of protein in biological fluids and interpreting of results of analysis in norm and pathology.To develop the proof of protein nature of enzymes.
Literature:
1. The tutorial book "Principles of biochemistry", 2005.p. 5-22
2. "Biochemistry", Pamela C. Champe at al.2005.p. 1-5, 13-21
3. The «Proteins», Lecture Materials;
Basic level tasks:
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1. Which one of the following statements concerning aspartate is correct?
A. It contains an amino group
B. It is classified as acidic amino acid in neutral solutions.
C. It is classified as basic amino acid in neutral solutions.
D. It has isoelectric form at pH 7,0.
E. It migrates to the cathode during electrophoresis in basic solution.
2. Which amino acid contains an amide group?
A. Alanin
B. Serine
C. Valine
D. Arginine
E. Glutamine
3. Which amino acid is hydrophobic?
A. Glutaminic acid.
B. Lysine
C. asparaginic acid
D. Cysteine
E. Methionine
4. Which amino acid can participate in disulphide bond formation?
A. Valine
B. Tryptophan
C. Cysteine
D. Serine
E. Glutamate.
5. Which amino acid has isoelectric form in neutral solution?
A. Alanine
B. Lysine
C. Hystidine
D. Glutamic acid.
E. Arginine
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The main theoretical questions:
1. Structure, physical and chemical properties, classification of amino acids.
2. Structure of peptides and peptide-bond characteristics.
3. Structural organization of protein molecules (primary, secondary, tertiary and quaternary structure)
4. Classification, physical and chemical properties of protein. (Charge, electrophoresis, denaturation)
5. The biological functions and role of proteins.
6. Proof of protein nature of enzymes.
Practice instructions:
Work 1. Quantitative definition of protein by a biuretic method.
The essence of the method: The method is based on ability of peptide bonds of protein to form in an alkaline conditions with ions of copper (Cu2+) complex of violet color, which intensity proportional to the contents of protein in medium.
Sequence of procedure:
1. Put 1 ml of protein solution (blood serum) in tube.
2. Add 1 ml 3% NaOH and 0, 1 ml of Benedict’s reagent.
3. Mix the content of tube good and leave for 15 min.
4. In 15 min. determine density of solution on photoelectrocolorimetre at a green light filter (wave length 540 nm.)
5. Protein concentration (in gram/l) determine from the standard curve.
6. By plotting density as ordinate versus concentration as abscissa is obtained standard curve. You can get this plot from technicians and should copy it's into followed axes:
Results:
Work 2. Proof of protein nature of enzymes.
The color reactions have been used to indicate the protein presence in solutions and establish amino acid composition of proteins.
There are 2 types color reaction:
1. Universal (biuret and ninhydrine)
2. Specific reaction for individual amino acids (for example, xantoprotein – for aromatic amino acids)
A. Biuret reaction: Sequence of procedure:
1. Put 3 tubes in support.
2. Pour 1 ml of ovoalbumin into the first tube, 1 ml of pepsin into the second tube and 1 ml of amylase solution into third tube.
3. Add 1 ml 3% NaOH and 0,2 ml Benedict solution into each of three tubes.
4. Mix well and observe the color. The appearance of violet color is a good evidence for the presence of a protein.
B. Ninhydrine reaction: Αmino acids (free and included in proteins and peptides) give complex compound of dark blue and blue –violet color with ninhydrine
Sequence of procedure:
1. Put 3 tubes in support.
2. Pour 1 ml of ovoalbumin into the first tube, 1 ml of pepsin into the second tube and 1 ml amylase solution into third tube.
3. Add 0,5 ml 0,5 ninhydrin solution into each of three tubes.
4. Boil solutions in the tubes 1-2 min carefully and observe the color.
The appearance of pink-violet color (which can become dark-blue in time) is a good evidence for the presence of a protein.
C. Xantoprotein reaction.
Aromatic amino acids (free and included in peptides and proteins) form compound of yellow color with concentrated nitric acid, which changes to orange by alkali addition.
Sequence of procedure:
1. Put 3 tubes in support.
2. Pour 1 ml of ovoalbumin into the first tube, 1 ml of pepsin into the second tube and 1 ml of amylase solution into third tube.
3. Add 5 drops of concentrated nitric acid into all 3 tubes.
4. Hit solutions carefully and observe the color.
5. Cool the tubes, add 0,5 ml of 20% NaOH and observe the change of color
Yellow color appeases and change to orange after expositing to cold and addition of NaOH if the solution contains an aromatic amino acid.
6. Put the date into the table.
№ of thetube / Research solution / Color of solutions in reaction
Biuret / Ninhydrine / Xantoprotein
1 / ovoalbumin
2 / pepsin
3 / amylase solution
Conclusions:
M. C. Q
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1. Which of the following reagents can denature proteins?
A. Trypsine
B. NaCl
C. Concentrated HCl
D. Glucose
E. 0,01 mol/l NaOH
2.Which one of the following statements concerning lysine is correct?
A.. .It contains an amino group in side chain.
B. It is classified as acidic amino acid in neutral solutions.
C. It is classified as basic amino acid in neutral solutions.
D. It has isoelectric form at pH 7,0.
E. It migrates to the anode during electrophoresis in basic solution.
3. Which one of the following statements concerning phenylalanine is correct?
A.. It contains an amino group in side chain.
B. It is classified as acidic amino acid in neutral solutions.
C.It is classified as basic amino acid in neutral solutions.
D. It is hydrophobic amino acid
5. It migrates to the anode during electrophoresis in neutral solution.
4. Which amino acid is hydrophobic?
A.. Glutamic aci 4.
B. Lysine
C. Aspartic acid
D. Cysteine
E. Methionine
5. Denaturation of protein is:
A.. The hydrolysis of peptide bonds
B. The cleavage of protein into small peptides
C.The cleavage of amino acids from N-terminal end of protein
D. Unfolding and disorganization of protein structure
E. The cleavage of amino acids from C-terminal end of protein
6. The primary structure of protein is stabilized by:
A. Hydrogen bonds
B. Peptide bonds
C.Hydrophobic interactions
D. Ionic interactions
E. Phosphoester bonds
7. The secondary structure of protein is stabilized by:
A. Phosphodiester bonds
B. Hydrophobic interactions
C.Covalent polar bonds
D. Hydrogen bonds
E. Ionic interactions
8. Which one of the following statements concerning primary structure of protein is correct?
A.. It is unique three-dimensional structur 5.
B. It is stabilized by hydrogen bonds.
C.It is stabilized by hydrophobic interactions
D. It is a sequence of amino acids binding by peptide bonds
5. It is a sequence of nucleotides.
9. Which one of the following statements concerning secondary structure of proteinis correct?
A.. It is unique three-dimensional structur 5.
B. It is stabilized by hydrogen bonds.
C.It is stabilized by hydrophobic interactions
D. It is a sequence of amino acids binding by peptide bonds
E. It is a sequence of nucleotides.
10. Which one of the following statements concerning tertiary structure of protein is correct?
A.. It is unique three-dimensional structur 5.
B. It is stabilized by ester bonds.
C. It is an arrangement of several polypeptide subunits into multimeric structur 5.
D. It is a sequence of amino acids binding by peptide bonds
E. It is a sequence of nucleotides.
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Topic 2.2. THE METHODICAL GUIDELINES FOR PRACTICE ACTIVITY ON THE THEME:
The investigation of enzymes structure, physical-chemical properties and conditions of action.
Biomedical importance:
Without enzymes, life as we know it would not be possible. As the biocatalysts that regulate the rates at which all physiologic processes take place, enzymes occupy central roles in health and disease. While in health all physiologic processes occur in an ordered, regulated manner and homeostasis is maintained, homeostasis can be profoundly disturbed in pathologic states. For example, the severe tissue injury that characterizes liver cirrhosis can profoundly impair the ability of cells to form the enzymes, which catalyze a key metabolic process such as urea synthesis. The resultant inability to convert toxic ammonia to nontoxic urea is then followed by ammonia intoxication and ultimately hepatic coma. A spectrum of rare but frequently debilitating and often fatal genetic diseases provides additional dramatic examples of the drastic physiologic consequences that can follow impairment of the activity of but a single enzyme.
Following severe tissue injury (eg, cardiac or lung infarct, crushed limb) or uncontrolled cell growth (eg, prostatic carcinoma), enzymes that may be unique to specific tissues are released into the blood. Measurement of these intracellular enzymes in blood serum therefore provides physicians with invaluable diagnostic and prognostic information.
The purpose: To develop skills in interpreting the properties and functions of enzymes for use this knowledge in diagnostics, prevention and treatment of diseases related to enzyme disorder.
Literature:
1. The tutorial book "Principles of biochemistry", 2005.p.39-41,43-44
2. "Biochemistry", Pamela C. Champe at al.2005.p. 53-58
3. Lecture on the theme «Enzymes»,
The main theoretical questions:
1. Definition and chemical nature of enzymes.
1.1. Proof of protein nature of enzymes;
1.2. Common and distinct features in enzymes and non enzymic catalysts.
2. Structural and functional organization of enzymes:
2.1. Simple and compound enzyme proteins. Cofactors, their nature and role in enzyme function;
2.2. Structure of an Active centre, its role in enzyme function;
2.3. Allosteric centre.
3. Properties of enzymes: