Qualitative identification of amino transfer reactions.

First, the experimental purpose
(1) to learn a simple method and principle to identify the role of amino transfer;
(2) to further understand the principles and operating techniques of paper analysis,
(3) to understand the significance of amino transfer in intermediate metabolism.
II, the experimental principle
amino transaminase is also known as
transaminase
, it can catalyz α-
amino acids
amino and α-ketoic acid α-ketone-ketone exchange, this effect is called amino transducation. It plays an important role in the synthesis and decomposition of
proteins
in organisms, and in the interconnement and transformation of sugar, fat and protein. When any amino acid performs transamination, it is catalyzed by its single-specific transaminase.
their most suitable pH is close to 7.4. Among all kinds of transaminases, glutamate-oxyacetic acid transaminase (glutamate transaminase, GOT) and glutamate-acetone transaminase (glutamate transaminase, GPT) are the most active.
III,
reagents
and equipment
anatomy equipment,
centrifuges
,
rthmostat
water bath,
drying
boxes, scales, layering systems, etc.
1/15mol/L phosphate buffer (pH=7.4), 1% glutamate solution (neutral to neutral with KOH), 1% acetone acid solution (medium to neutral with KOH), 0.1% potassium bicarbonate solution, 0.05% iodine acetic acid solution, 15% triclosan acetic acid solution, standard alanine solution (0.1%), standard glutamate solution (0.1%), 0.1% hydration
, the operation method
the rabbit will be knocked out, quickly dissected, removed the liver, cut in low temperature conditions. After weighing the slurry, centrifugation is hydrate, i.e. the preparation of the enzyme solution. In-body amino shift reaction. Paper on-paper subsolution method to check each person 4 points (standard alanine solution, standard glutamate solution, control on the liquid, sample on the liquid).
point sample into the laminate cover. With an aqueous solution saturated with phenol, after balancing, the phenol reagent is added 25 ml from the laminate cover with a glass tube with a small funnel. Then, immediately cover the plug, when the solvent front reaches about 2.5cm from the top end of the filter paper, remove the filter paper, dry or blow dry, to remove the phenol solvent, spray 0.1%tritone ethanol solution, put in the oven to show the tryptophan andtri ketone reaction, on the filter paper showing purple spots.
, key steps and precautions
(1) take animal fresh
tissue
preparation enzyme solution, to prevent enzyme infestion.
(2) iodized acetic acid inhibits enzymatic dissophation.
.