The transamination of transaminase was identified by paper-based analysis.

basic principles
the role of
-trans-amino is widely found in the body's
tissues
organs, is an important way of metabolism of
amino acids
in the body. Amino acid reactions are catalyzed by a single-specific
-transaminase
, which catalyses the α-amino of amino acids to another α-ketone acid. The activity of various transaminases is different, among which the liver's Glutamic Pyruvic Transaminase (GPT) is more active, which catalytics the following reactions:
α-ketone diacid and alanine Glutamate and acetone
This experiment with alanine and α-ketone diacine as the substrate, plus liver homogeneum insulation, paper layering method to check the appearance of glutamate to prove the role of transaminine.
paper layering belongs to the distribution layering. With filter paper as the support, filter paper fiber and water affinity are strong, can absorb 20 to 22 percent of the water. And 6 to 7 percent of the water is in the form of hydrogen bonds and cellulose hydroxyl binding, in general it is difficult to take off, and filter paper fiber and
organic
solvent affinity is very weak. Therefore, paper lamination is based on filter paper fiber binding water as a fixed phase, organic solvent as a flow phase.
the paper layering method to separate amino acids, due to the various amino acids in this two phases of the distribution coefficient is different, each has a certain migration rate. The amino acids with weak polarity are easily soluble in organic solvents, i.e. the distribution coefficient is small, the flow phase moves faster, the rf value is large, and the extreme amino acids are opposite, according to which the amino acids can be isolated and identified.
experiment with round filter paper for horizontal layering, in addition to the liquid to be measured, point plus control liquid and standard liquid, after the layering withtritone method color, to observe the color spot, determine the results.equipment
glass homogenizer, centrifugal tube, 10 ml test tube,
culture
dishes, surface dishes, boiling water bath pot, 37 degrees C
string temperature
water bath tank, 10cm round filter paper, hairdryer, surgical scissors.Reagent
Reagents
1.
1. 0.01mol/L pH 7.4 phosphate buffer.
2. 0.2mol/L Na2HPO4 solution 81ml mixed with 0.2mol/L NaH2PO4 solution 19ml, diluted 20 times with distilled water.
3. 0.1mol/L Alanine solution
is said to take 0.891 grams of alanine, first dissolved in a small amount of 0.01mol/L pH 7.4 phosphate buffer, with 1.0 N NaOH carefully adjusted to pH7.4, add phosphate buffer to 100ml.
4. 0.1mol/L alpha-ketone diacid
is called α-ketone diacid 1.461 g, first dissolved in a small amount of 0.01mol/L pH 7.4 phosphate buffer, with 1.0 N NaOH carefully adjusted to pH 7.4, add phosphate buffer to 100ml.
5. 0.1mol/L glutamate solution
is called glutamate 0.735 grams, first dissolved in a small amount of 0.01mol/LpH 7.4 phosphate buffer, with 1.0 N NaOH carefully adjusted to pH 7.4, add phosphate buffer to 50 ml.
6. 0.5 tritone solution
0.5 g ofis said to dissolve in 100 ml of acetone.
7. Layering solvent
: water: 4:1 (V/V) mixed spare. Ready.Basic Operations
1. Homogenous preparation: take 0.5 grams of fresh animal liver, cut and put into the slurryer, add cold 0.01mol/LpH 7.4 phosphate buffer 1.0 ml, quickly developed into a homogenous slurry, with the above buffer 3.5 ml mixed spare.
2. Enzyme reaction process
(1) take two centrifugal tubes, number: 1 (assay tube), 2 (control tube), each plus liver plasma 0.5 ml. Put the control tube in a boiling water bath and heat
for
5 minutes before removing and cooling.
(2) plus 0.1mol/L alanine 0.5 ml, 0.1mol/L alpha-ketone diacid 0.5 ml, 0.01mol/L pH 7.4 phosphate buffer 1.5 ml, shake well.
(3) 37 degrees C to keep warm and remove after 1 hour.
.