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) -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 reduced forms of nicotinamide and flavine oxidoreductases.

Pour 0.5 ml of water and 1 drop of riboflavin solution into a test tube. Add some drops of methylene blue till the mixture turns green-blue. Throw a piece of zinc into the colored mixture and add 2 drops of concentrated hydrochloric acid.

Hydrogen is released/ It decreases redox potential of the system, and methylene blue and riboflavin are reduced. How the color of mixture is changed? What component of the solution is reduced faster?

Pour the liquid into another test tube and observe the color changes. Now hydrogen is not formed, but released from the solution into air. The reduced form of riboflavin transmits electrons and ions of hydrogen through methylene blue to aerial oxygen. After this leucomethylene blue is oxidized.

Identifying of catalase activity (method of A.N. Bach and А.I. Oparin).

Take water extract of carrots which contains the enzyme catalase for identifying. 100 ml of it is prepared from 2 g of fresh carrot.

Pipette 20 ml of enzyme extract into two conical flasks. Put one of flasks in the boiling water bath for 5 minutes (control sample, enzyme inactivation), then cool it.

Then pipette 25 ml of 0,1 n hydrogen peroxide solution in both test tubes. In 30 minutes stop the action of the enzyme by adding 5 ml of 10% sulfuric acid solution and titrate mixtures by 0.1 n. potassium permanganate solution (till you see the fixed pink coloring during approximately 1 minute). Mark volumes of potassium permanganate solution used for titration of hydrogen peroxide in control and experimental samples.

Calculate enzyme activity in standard international units (U) according to the reaction equation:

5Н₂О₂ + 2КМnО₄ + 3H₃SO₄ ® 2MnSO₄ + K₂SO₄ + 5О₂ + 8Н₂О

by the formula:

, where:

V_c – volume of 0.1 npotassium permanganate solution used for the titration of control sample, ml;

V_ex – volume of 0.1 npotassium permanganate solution used for the titration of experimental sample, ml;

100 – volume of carrot extract;

1.7 – mass of hydrogen peroxide corresponding to 1 ml of 0.1 npotassium permanganate solution, mg

20 – volume of carrot extract taken for analysis, ml;

2 – carrot mass, g

30 – time of experiment, min;

0.034 – mass of 1 micromole of hydrogen peroxide, mg.

Tasks

1. Give the Enzyme Commission number for enzymes used in the labwork..

2. For experiment 3 make up a scheme of hydrogen transfer taking into account standard oxidation-reduction potentials of the used components.

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