9.2. Reaction with methylene-blue.
Pour 2 drops of 10% methylene-blue solution, 2 drops of 10% sodium hydrocarbonate solution, 10 drops of vitamin C solution into a test tube and heat. The liquid becomes transparent.
Test questions
1. What substanses belong to vitamins? What is their general function in an organism?
2. List the biochemical functions of the vitamins which were identified in the laboratory work.
3. Define the terms: avitaminosis, hypovitaminosis and hypervitaminosis.
Laboratory work 7. Oxidoreductases. Common catabolic pathway
1. Comparison of redox-potentials of riboflavin and methylene blue.
The redox-potentials are the measure of molecules’ ability to exchange electrons. The substance with high redox-potential oxidizes the substance with less redox-potential. For methylene blue (±2 е) Е'о is +0,011 V, for NAD+ (±2 е) - 0,320 V, for riboflavin (±2 е) - 0,208 V, for cytochrome с (±е) + 0,260 V. Comparing standard redox-potentials of these systems, one can make a conclusion, that methylene blue can be used for identifying the redused forms of nicotinamide and flavine oxidoreductases.
Pour 5 - 6 drops of water, 1 drop of riboflavin solution into a test tube. Add drops of methylene blue till the mixture turns green-blue. Throw a piece of zinc into the colored mixture and add a drop of concentrated hydrochloric acid.
Hydrogen is released and methylene blue and riboflavin are reduced. The first of them is reduced faster, that is why the color of the mixture first turns green, then – green-yellow and finally – pale-yellow or pinkish. Pour the liquid into another test tube and observe the color changes. The reduced form of riboflavin transmits electrons and ions of hydrogen through methylene blue to aerial oxygen, and the solution turns yellow. After this leucomethylene blue oxidizes and the contents of the test tube changes color through green into blue.
2. Identifying of catalase activity according to a.N. Bach and а.I. Oparin.
Take water extract of carrots which contains the enzyme catalase for identifying. Pipette 25 ml of 0,1 n hydrogen peroxide solution into a conical flask. Then pipette 20 ml of enzyme extract there. In 30 minutes stop the action of the enzyme by adding 5 ml of 10% sulphuric acid solution and titrate the mixture by 0,1 n. potassium permanganate solution (till you see the fixed pink coloring during approximately 1 minute).
Simultaneously put control with inactivated warming in the boiled water bath for 5 minutes enzyme solution (20 ml.). After cooling add 25 ml of 0,1 n hydrogen peroxide solution to this solution. Leave the mixture for 30 minutes and then add 5 ml of 10% sulphuric acid solution and titrate by 0,1 n potassium permanganate solution. By difference between the test and control titrating identify the quantity of permanganate, equivalent to the quantity of the hydrogen peroxide decomposed by the enzyme.
The quantity of hydrogen peroxide, decomposed by the enzyme is counted according to the reaction equation:
5Н2О2 + 2КМnО4 + 3H3SO4 2MnSO4 + K2SO4 + 5О2 + 8Н2О,
according to which 1 ml of 0,1 n potassium permanganate solution corresponds to 1,7 mg of hydrogen peroxide.
An
example of calculation: 100 ml of catalase extract is prepared from
1,25 g of carrots. 15,5 ml are used for experiment test titrating,
30,2 ml of 0,1 n
potassium
permanganate solution are used for control test titrating. The amount
of the decomposed
hydrogen
peroxide in the test is equivalent (30,2 - 15,5) 14,7 ml. 0,1 n
potassium permanganate solution and therefore, is equal (14,7 - 1,7)
24,99 mg 1 g of raw carrots contains the amount of catalase, able to
decompose
99,96 mg
of hydrogen peroxide for 30 minutes, and (99,96:30)
3,33 mg – for a minute. As 1 mcmole of hydrogen
peroxide is 0,034
mg, 1 g of carrots contains (3,33:0,034)100 U of catalase.
Experiment 2. Quantitative determination of pyruvic acid in urine.
Pyruvic acid is one of the intermediate products of carbohydrate metabolism. Under anaerobic conditions (hypoxia) it is reduced into lactic acid, and under aerobic ones it undergoes oxidative decarboxylation and is converted into acetyl-coenzyme A. Pyruvic acid is one of the main sources for gluconeogenesis. As a result of high rate of the conversion pyruvic acid is present in tissues and biologic liquids in little amounts. In blood its amount is 0,5-1 mg/100 ml. The amount of pyruvate in urine is normally 2 mg/100 ml, its daily excretion with urine is - 10-25 mg.
The most rapid increase of pyruvate concentration in blood and as a consequence in urine is noticed during muscular work and В1 vitamin deficiency. This phenomenon is also noticed during hepatic disorders, diabetes, cardiac decompensation, toxicosis, etc.
The method principle: pyruvate and 2,4-dinitrophenylhydrazine form a colored compound 2,4-dinitrophenylhydrazone of pyruvate. It is extracted from the reaction mixture by toluene. When alkali alcohol solution is added, it gradually turns red-orange; the optical density of the solution is directly proportional to the quantity of pyruvic acid.
pyruvic acid 2,4-dinitrophenylhydrazine dinitrophenylhydrazone of pyruvic acid
Course of work: Pour 0,5 ml of 0,1% 2,4- dinitrophenylhydrazine up to 0,5 ml of urine, mix and add 2,5 ml of water-saturated toluene after 5 minutes. After shaking for 1 minute leave the solution for water and toluene demixing. Take a 1 ml test from the upper toluene layer with a dry graduated pipette and place it into a dry test tube, add 3 ml of potassium hydroxide alcohol solution. Process the test with 1 ml of standard solution of pyruvate in the same way as the experiment test. In the chemical reagents control the urine is substituted by 1 ml of water. After 10 minutes photometer the tests against the chemical reagents control with the wave length of 400-415 nm using photoelectric colorimeter.
The calculation is made according to the formula: Х=(De*50mg)/Ds, where: De – is optical density of the experimental solution, Ds – is optical density of the standard solution, 50 mg – is the concentration of pyruvic acid standard, Х – is the concentration of pyruvate in urine.