Test questions
1. What are activators and inhibitors of enzymes?
2. How can the influence of activators and inhibitors upon the work of enzymes be investigated?
3. The enzyme acetylcholinesterase catalyses the hydrolysis of acetylcholine. It is neuromediator produced in the synapses of cholinergic nerves. Its breakdown products - acetate and choline – are unable to work as neuromediators. The hydrolysis of acetylcholine is stopped by calimine – a medicinal preparation used to heal motor disturbance after injuries, paralysis, in rehabilitating periods after having poliomyelitis, encephalitis, etc. The following scheme reflects the inhibition of acetylcholinesterase:
Inhibitor calimine
acetylcholine choline acetic acid
Compare the structural formulas of the inhibitor and the substrate. Why can one suppose that the inhibitor is bounding in the active centre of the enzyme? How will the influence of calimine change the conduction of nerve impulse (increase, reduce or will not change)?
Laboratory work 6. Vitamins
Experiment 1. Qualitative reactions to vitamin A (retinol).
1.1. Reaction with antimony chloride (III).
Place 1-2 drops of fish liver oil or solution of vitamin A in oil into a dry test tube, add 5-6 drops of antimony chloride (III) solution in chloroform and 2-3 drops of acetic anhydride. You will see blue coloring gradually turning pink-violet. The reaction is unspecific as it can be also given by some other compounds having conjugated double bonds.
1.2. Reaction with sulfuric acid (Drummond’s reaction).
Dissolve 1 drop of fish liver oil (or vitamin A) in 4 drops of chloroform and add 1-2 drops of strong sulfuric acid. You will see blue coloring immediately turning brown-red.
The given reactions are based on the dehydrating influence of antimony chloride (III) and sulfuric acid on vitamin A.
Vitamin a (vitamin a, retinol)
Experiment 2. Qualitative reactions to vitamin D (calciferol).
2.1. Reaction with bromine solution in chloroform.
Mix 0,5 ml of fish liver oil and 0,5 ml of bromine solution in chloroform in a dry test tube. The presence of vitamin D will give green-blue coloring. The reaction is unspecific.
2.2. Reaction with aniline.
Pour 0,5 ml of fish liver oil and 0,5 ml of aniline and strong sulfuric acid mixture into a dry test tube (15:1), mix properly, heat carefully up to boiling for a minute. If vitamin D is present, the color of this emulsion will be green and then - red. Leave the test tube at the room temperature. The emulsion is layered; the lower layer is turning bright-red.
Vitamin d (calciferol)
Experiment 3. Qualitative reactions to vitamin E (tocopherol).
3.1. Reaction with concentrated nitric acid.
Add 0,5 ml. of concentrated nitric acid to 5-6 drops of 0,1% spirit tocopherol solution in a dry test tube and shake. You will get a gradually layered emulsion the upper layer of which is red. The coloring is caused by tocopherol oxidation.
-tocopherol -tocohinon (toco-red)