First, the purpose of the experiment
(1) Learning a simple method for identifying aminotransfer and its principle;
(2) further master the principle and operation technology of paper chromatography;
(3) Understand the significance of aminotransfer in intermediate metabolism.
Second, the experimental principle
Aminotransferase, also known as transaminase, catalyzes the exchange of the amino group of an alpha-amino acid with the alpha-keto group of an alpha-keto acid. This action is known as aminotransfer. It plays an important role in the intermediate metabolism of protein synthesis and decomposition in organisms, in the interaction and mutual transformation of sugar, fat and protein metabolism. Any amino acid undergoing transamination is catalyzed by its specific transaminase. Their optimum pH is close to 7.4. Among various transaminase, glutamic acid-oxaloacetate transaminase (referred to as aspartate aminotransferase, GOT) and glutamate-pyruvate transaminase (abbreviated as alanine aminotransferase, GPT) have the strongest activity.
Third, reagents and equipment
Anatomical instruments, centrifuges, constant temperature water baths, drying ovens, scales, chromatography systems, etc.
1/15mol/L phosphate buffer (pH=7.4), 1% glutamic acid solution (neutralized to neutral with KOH), 1% pyruvic acid solution (neutralized to neutral with KOH), 0.1% potassium bicarbonate Solution, 0.05% iodoacetic acid solution, 15% trichloroacetic acid solution, standard alanine solution (0.1%), standard glutamic acid solution (0.1%), 0.1% ninhydrin ethanol solution, phenol solvent.
Fourth, the operation method
The rabbit was stunned, dissected quickly, and the liver was removed and chopped under low temperature conditions. After the homogenate is weighed, the supernatant is centrifuged to obtain the prepared enzyme solution. In vitro aminotransfer reaction. Four points per person (standard alanine solution, standard glutamic acid solution, control supernatant, sample supernatant) were examined by on-paper chromatography. Place the sample in the chromatographic hood. After equilibration with an aqueous solution saturated with phenol, 25 ml of a phenol reagent was added to the chromatography chamber from a chromatographic flask using a glass tube with a small funnel. Then, immediately close the plug, when the solvent front reaches about 2.5cm from the upper end of the filter paper, take out the filter paper, air dry or blow dry to remove the phenol solvent, spray 0.1% ninhydrin ethanol solution, put it into the oven to develop color amino acids and The ninhydrin reaction exhibited a purple-red spot on the filter paper.
Five, key steps and precautions
1 Take the fresh tissue of the animal to prepare the enzyme solution to prevent the enzyme from inactivating.
2 iodoacetic acid inhibits enzymatic hydrolysis.
Amniotic Membrane Perforator,Amniotomy Hook,Medical Amniotomy Hook,Sterile Amniotomy Hook,Amniotic Fluid Hook,Amniotic Hook, Amnihook, Amnio hook
Amniotomy Hook,Amniotic Membrane Perforator ,Amnihook, Amniotic Fluid Hook,Amniotic Hook
Luck Medical Consumables Co.,LIMITED , https://www.luckmedical.com