Methodical Instruction for Students of the 1 Course Medical Faculty
LESSON № 5 (PRACTICAL – 6 HOURS)
Themes: 1. Amino acids as structural components of properties and peptide. Chromatographic separation of amino acids.
2. Hydrolysis of simple proteins. Precipitation reactions of proteins.
3. The determination of the protein contents in blood serum.
Aim: To be able to determine amino acids in solution by qualitative tests; to explain their chemical properties; to analyze the amino acid composition of solution using the partition filter-paper chromatography and use this method for diagnostic purpose; to carry out the reversible and inreversible precipitation of protein; to determine the total contents of proteins in blood serum and explain causes and consequences of the deviation of this indicator from normal level.
Professional orientation of students:
Since proteins are fundamental in all aspects of cell structure and function the knowledge of their chemical composition is necessary for future doctors to diagnose and treat various diseases, specifically congenital diseases. Knowledge of the reactions of amino acids is useful in several important aspects of protein chemistry: (1) identification and analysis of amino acids in protein hydrolyzates, (2) identification of the amino acid sequence in protein molecules, (3) identification of the specific amino acid residues of native proteins that are required for their biological function, (4) chemical modification of amino acid residues in protein molecules to produce changes in their biological activity or other properties, and (5) chemical synthesis of polypeptides.
Methodology of Practical Class (900-1200).
Investigation of structure, physical and chemical properties of aminoacids, protein, peptides. Mechanism of nucleophilic addition reactions and nucleophilic substitution reactions.
Theme 1. Amino acids as structural components of properties and peptide. Chromatographic separation of amino acids (2 hours).
Work 1. Ninhydrin reaction.
On heating an a-amino acids react with two molecules of ninhydrin to yield an intensely violet colored product. A purple color is given in the ninhydrin reaction by all amino acids and peptides having a free a-amino group.
Materials. 1 % solution of glycine, 0,1 % solution of ninhydrin,
Protocol. Add in the tube:
4 drops solution of glycine,
2 drops of 0,1 % solution of ninhydrin.
Solution in the tube is warmed, after that solution in tube can be violet colour.
Work 2. Detection carboxylic groups in glycine.
Materials. 1 % solution of glycine, 40% solution of formaldehyde, 0,2% solution of methylred.
Protocol. Add in the tube:
5 drops 1 % solution of glycine,
1 drops of solution of 0,2% solution of methylred
solution in tube can be yellow colour (neutral medium pH=7);
add 5 drops 40% solution of formaldehyde
solution in tube can be red colour (acid medium pH <7)
This reacting will be used for quantitative determination carboxylic groups in a-amino acids.
Work 3. Detection amino groups in glycine.
Materials. 1 % solution of glycine, 1% solution of NaNO2, acetic acid.
Protocol. Add in the tube:
5 drops 1 % solution of glycine,
5 drops 1 % solution of NaNO2,
2 drops acetic acid
The out gassing (N2) is watched. Reacting will use for quantitative determination amino groups in amino acids.
Work 4. Xanthoprotein reaction.
This reaction detects the aromatic amino acids (phenylalanine, tryptophan, tyrosine). In action of the nitric acid aromatic rings of the amino acids are nitrated to yield yellow compounds. Yellow color is transformed to orange in ammonia adding.
Materials. 1 % solution of thyrosine, solution of HNO3,
Protocol. Add in the tube:
5 drops 1 % solution of thyrosine,
1 drops of solution of HNO3.
Solution in the tube is warmed, after that solution in tube can be yellow colour.
If after some minutes (3-5) 10 drops NH4OH (ammonia) solution was added in the tube, so solution’s colour is changed to orange colour.
Work 5. Foll reaction.
This reaction reveals the sulfur containing amino acids (cysteine, cystine). Treatment of the sulfur containing amino acids with salt of lead and alkali yields a black sediment.
Materials. Solution of protein, 5 % solution of lead acetate, 30 % NaOH.
Protocol. Add in the test-tube: 5 drops of the solution of cysteine and cystine or protein 5 drops of the 30 % NaOH; drop of the 5 % solution of lead acetate ((CH3COO)2Pb).
Result. The black color is formed.
Work 6. Adamkevich reaction.
This reaction detects the amino acid tryptophan containing indol ring. The addition of the concentrated acetic and sulfuric acids to the solution of tryptophan results in the formation of red-violet ring appearing on the boundary of different liquids.
Materials. Solution of protein, concentrated acetic and sulfuric acids.
Protocol. Add in the test-tube: 2-3 drops of the solution of tryptophan or protein.
1-2 drops of the concentrated acetic acid
2-3 drops of the concentrated sulfuric acid
(add carefully on the wall of test-tube).
Result. The red-violet ring is formed on the boundary of different liquids.
Work 7. Qualitative reactions on proteins Biuret reaction.
Treatment of a peptide or protein with Cu2+ and alkali yields a purple Cu2+-peptide complex.
Materials. Protein solution, 10 % NaOH, 1 % copper sulfate.
Protocol. Add in the test-tube: 2-3 drops of the protein solution
1-2 drops of 10 % NaOH
1 drop of 1 % copper sulfate.
Result. The purple color is formed.
Theme: 2. Hydrolysis of simple proteins. Precipitation reactions of proteins (2 hours).
Work 8. Salting-out of proteins.
The concentrated solution of alkaly metal salts react with protein molecule and as result the dehydratation of proteins, thus reducing their solubility and precipitation.
Materials. Blood serum, saturated solution of ammonium sulfate, powder of ammonium sulfate, 10 % solution of NaOH, 1 % solution of copper sulfate.
Protocol. Add in the tube: 2-3 ml of blood serum the same volume of saturated solution of ammonium sulfate (50 % saturation);
globulins are precipitated;
filter;
add to filtrate powder of ammonium sulfate
until salt stop to solve;
observe the precipitation of albumins;
filter;
carry out the biuret reaction with filtrate.
Work 9. Denaturation of proteins.
Proteins can be denaturated in the acting of salts of heavy metals, high temperature, concentrated acids and alkalines, alkaloids. Salts of heavy metals form unsoluble complex compounds with proteins; in acting of temperature, concentrated acids and alkalines, alkaloids the non-covalent bonds break down. As result the denaturation takes place.
Materials. 1 % solution of egg white, 5 % solution of copper sulfate, 5 % solution of lead acetate, 5 % solution of silver nitrate, concentrated nitric, sulfosalicylic and trichloracetic acids, 10 % solution of picric acid, saturated solution of tanin, 5 % solution of K3Fe(CN)6.
Protocol. Add into the 10 test-tubes: 1 ml of 1 % solution of egg white;
Add into the 1-st tube 3 drops of 5 % solution of copper sulfate;
2-nd – 3 drops of 5 % solution of lead acetate;
3-d – 3 drops of 5 % solution of silver nitrate;
4-th – 3 drops of concentrated nitric acid;
5-th – 3 drops of concentrated sulfosalicylic acid;
6-th – 3 drops of concentrated trichloracetic acid;
7-th – 3 drops of 10 % solution of picric acid;
8-th – 3 drops of saturated solution of tanin;
9-th – 3 drops of 5 % solution of K3Fe(CN)6;
Heat 10-th tube to boiling.
Observe the denaturation of proteins in all tubes.
Theme 3. The determination of the protein contents in blood serum.
Work 10. The determination of protein contents in blood plasma by the biuret method.
Interplay of the peptide bonds with copper sulfate in alkaline medium results in the purple color formation. The intensity of the dyeing is straightly proportional to the protein concentration and is determined by the colorimetric method.
Materials. Blood plasma, standard protein solution (50 g/l), biuret reagent.
Protocol. Add in the 1-st tube 0,1 ml of blood plasma, in the 2-nd – 0,1 ml of the standard protein solution and in the 3-rd – 0,1 ml 0,9 % NaCl (control). Add in each tube on 5 ml of the biuret reagent, mix and determine the extinction after 30 min. As control use the 3-rd tube. For the analysis apply the red light filter (750 nm). For the calculation use the following formula: X = A x B/C.
X – protein contents in studied sample;
A – extinction of the sample with blood plasma;
B – protein concentration in standard solution;
C – extinction of the sample with standard protein solution.
Work 11. The determination of protein contents in blood plasma by the refractometric method.
Materials. Blood plasma, refractometr.
Protocol. Add in the refractometr glass some drops of blood plasma and observe index of refraction.
|
Index of refraction |
Contents in blood plasma (g/l) |
Index of refraction |
Contents in blood plasma (g/l) |
Index of refraction |
Contents in blood plasma (g/l) |
|
1,34124 |
30,6 |
1,34537 |
54,7 |
1,34947 |
78,5 |
|
1,34162 |
32,8 |
1,34575 |
56,8 |
1,34984 |
80,5 |
|
1,34199 |
35,0 |
1,34612 |
59,0 |
1,35024 |
82,8 |
|
1,34237 |
37,2 |
1,34650 |
61,2 |
1,35058 |
84,9 |
|
1,34275 |
39,4 |
1,34687 |
63,4 |
1,35095 |
87,1 |
|
1,34313 |
41,6 |
1,34724 |
65,5 |
1,35132 |
89,2 |
|
1,34350 |
43,8 |
1,34761 |
67,7 |
1,35169 |
91,4 |
|
1,34338 |
46,0 |
1,34798 |
69,8 |
1,35205 |
93,5 |
|
1,34426 |
48,1 |
1,34836 |
72,0 |
1,35242 |
95,7 |
|
1,34463 |
50,3 |
1,34873 |
74,2 |
1,35279 |
97,8 |
|
1,34500 |
52,3 |
1,34910 |
76,3 |
|
|
Individual Students Program.
I. Amino acids as structural components of properties and peptide. Chromatographic separation of amino acids.
1. Biological role of amino acids.
2. Classification of amino acids.
3. Structure of amino acids. An amphoteric character they.
4. Chemical properties of amino acids:
a) Decarboxylation
b) Deamination
c) React with acids
d) React with the bases
5. Chemical composition of proteins.
6. Amino acids as the building blocks of proteins.
7. The structure and classification of amino acids.
8. The properties of amino acids.
9. Peptide bond – the basis of the protein molecule.
10. Qualitative reactions on the proteins and amino acids (biuret reaction, ninhydrin, Adamkevich, xanthoprotein, Foll and Millon reactions, determination carboxylic and amino-groups in molecule amino acids.). They’re practical using.
11. The general principle of chromatographic analysis of proteins and amino acids.
12. Kinds of chromatographies (partition, ion-exchange chromatography, gel-filtration, selective adsorption, affinity chromatography).
13. Specific and personal specificity of proteins.
14. Diagnostic significance of blood and urine chromatographic analysis. Hypo- and hyperaminoacidemia, hypo- and hyperaminoaciduria.
II. Hydrolysis of simple proteins. Precipitation reactions of proteins.
1. Structural organization of proteins.
2. Primary structure of proteins, methods of its determination (Sanger method, Edman method, specific hydrolysis).
3. To be able to write down the amino acids and polipeptides.
4. Secondary, tertiary and quaternary structures of proteins (definition, examples).
5. Types of bonds stabilizing the native structure of the protein.
6. The methods of determination of the secondary, tertiary and quaternary structures of proteins.
7. The effect chemical and physical factors (pH, temperature and radiation) on the structure and properties of proteins.
8. Properties of proteins.
9. The molecular weight of proteins and its determination.
10. Proteins solubility. Factors determining the solubility.
11. Methods of protein precipitation.
12. Reversible coagulation of proteins. Salting-out of proteins.
13. Separation, purification and characterization of proteins.
14. Protein denaturation and renaturation.
15. Using of protein capacity to salting-out and denaturation in medical practice.
III. The determination of the protein contents in blood serum.
1. The total contents of proteins in blood plasma.
2. The main proteins of blood plasma. Their physical and chemical properties and functions.
3. Causes and consequences of the change of total protein contents and its fractions in blood plasma.
4. The principles of biuret and refractometric methods of total protein determination in blood plasma.
Seminar discussion of theoretical issues (1230– 1400 hour).
Test evaluation and situational tasks.
1. The studying solution gives the positive ninhydrin and Foll reactions. Which component contains this solution?
2. The studying solution gives the positive biuret and xanthoprotein reactions. Which components this solution contains?
3. Which amino acid is in solution, if this solution has positive xanthoproteic reaction?
4. Solution, which is in the tube, has the positive ninhydrin test. What amino acids are in solution?
5. Which reaction gives the possibility to find the sulfur contained amino acids?
A. Ninhydrin
B. Adamkevich
C. Foll
D. Xanthoprotein
E. Millon
6. Which reaction gives the possibility to discover all a-amino acids?
A. Ninhydrin
B. Adamkevich
C. Foll
D. Xanthoprotein
E. Millon
7. Which color is yielded in the ninhydrin reaction?
A. Purple
B. Red
C. Green
D. Yellow
E. Blue
8. Which color is yielded in the biuret reaction?
A. Purple
B. Red
C. Green
D. Yellow
E. Blue
9. Which reagent is necessary for biuret reaction?
A. H2SO4
B. HCl
C. NaOH
D. Uric acid
E. Amino acid
10. What products is formed after heating of β-aminobutanoic acid:
A.
B.
C.
D.
E.
11. What amino acids are heterocyclic:
A. Lysine
B. Arginine
C. Histidine
D. Threonine
E. Methionine
12. The primary structure of protein is formed by such bond:
A. Ester;
B. Ionic;
C. Glycosidic;
D. Peptide;
E. Hydrogen.
13. Salting up is the precipitation of proteins in solution by:
A. Salts of heavy metals
B. Concentrated mineral acids
C. Saturated and half saturated solution of alkaline and alkaline-earth metals.
D. Alkaloids
E. High temperature
14. In which method solute molecules are sorted out by the differences in their acid-base behavior?
A. ion-exchange chromatography;
B. gel-filtration;
C. selective adsorption;
D. affinity chromatography.
15. Which method is based on a biological property of some substances for specific, noncovalent binding of another molecule, called the ligand
A. ion-exchange chromatography;
B. gel-filtration;
C. Selective adsorption;
D. affinity chromatography.
16. What level of amino acids in blood plasma iormal conditions?
A. 21,2 mmol/l;
B. 31,2 mmol/l;
C. 41,2 mmol/l;
D. 51,2 mmol/l.
E. 11.2 mmol/l.
17. Which of these methods can be used for measurement of protein moleculat weight?
A. biuret reaction;
B. sedimentation analysis;
C. ion-exchange chromatography;
D. polarographic.
18. Protein solubility depend on:
A. Osmotic pressure;
B. Shape of molecule;
C. pH;
D. Conformation of protein molecule.
19. How much albumins are in blood plasma of healthy people?
A. 40-50 g/l;
B. 50-60 g/l;
C. 30-40 g/l;
D. 35 –45 g/l;
E. 25-40 g/l.
20. How much globulins are in blood plasma of healthy people?
A. 35-45 g/l;
B. 30-40 g/l;
C. 25-35 g/l;
D. 45-55 g/l;
E. 10-20 g/l.
Correct answers of test evaluations and situational tasks:
1. a-Amino acids, cysteine, cystine.
2. Protein or peptides contained the aromatic amino acid.
3. Aromatic amino acids (phenylalanine, tyrosine, histidine).
4. a– Amino acids.
5. C; 6. A; 7. A; 8. A; 9. C; 10. E; 11. C; 12. D; 13. C; 14. A; 15. D; 16. A; 17. B. 18. C; 19. A; 20. B.
Individual student work (1415-1500 hour) are checked by solving situational tasks for each topic, answers in test evaluations and constructive questions (the instructor has tests & situational tasks).
Students must know:
– The biological role and chemical properties of amino aced;
– Structure and properties of amino aced.
– The kinds of chromatographies and interpretation of amino acid measurement in blood plasma and urine.
– The properties of proteins and the methods of their purification and separation;
– The contents of proteins in blood plasma as well as the causes and consequences of the change of total protein contents and its fractions.
Students should be able to:
– Determine the amino aced by qualitative test in solution.
– Perform the partition filter paper chromatography.
– Perform the precipitation of proteins by salting-out and denaturation;
– Determine the protein concentration in blood plasma.
References:
1. David Gutsche C., Baniel J. Pasto. Fundamentals of Organic Chemistry. – New Jersey: Prentice -Hall, Inc.Englewood Cliffs, 2005. -1346 p.
2. Joln W. Suttie. Introduction to Biochemistry. – New York: Holt, Rinehart and Winston, Inc., 2002. – 364 p.
3. Stryer L. Biochemistry. – New York: W.H. Freeman and Company, 2008. – 1086 p.
4. Lehninger A. Principles of Biochemistry. – New York: Worth Publishers, Inc., 2002. – 1010 p.
5 http://intranet.tdmu.edu.ua/data/kafedra/internal/zag_him/classes_stud/biological and bioorganic chemistry/medical/1 course/05. Amino acids. Determine total protein concentration in blood serum
Additional:
1. Andrew Streltwieser, Jr. Clayton H. Hcathcocr. Introduction to Organic Chemistry. – New York: Macmillan Publishinc Co., 2006. – 1508 p.
2. Lewis D. E. Organic chemistry. A modern perspective. – Copyright, 2006. P. 25 – 95
The methodical instruction has been worked out by: Associate Prof. Dmukhulska Ye. B.
Methodical instruction was discussed and adopted at the Department sitting
25.06.2013. Minutes N 11
Methodical instruction was adopted and reviewed at the Department sitting
27.08.2013. Minutes N 1
