The vitality of lactic acid dehydrogenase was determined.

related topicsthe(1) to understand the principle of
dehydrogenase
dehydrogenation.(2) learn how to determine enzyme activity by color ratio.Experimental PrinciplesLactate dehydrogenase (LACTate deogenhydrase LDH, EC.1.1.1.27, L-Lactic Acid: NAD-Redoxase) is widely present in biological cells and is one of the key enzymes in the enzymes of sugar metabolic enzymeation, catalyzing the following reversible reactions.. LDH is soluble in water or salt solutions.
the
LDH content measurement methods in the tissue, of which the UV terolight method is simpler and faster. In view of NADH, NAD plus at 340nm and 260nm have their own maximum absorption peak, so NAD plus as coenzyme of various dehydrogenase classes can be through 340nm light absorption value changes, quantitative determination of enzyme vitality, such as apple acid dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glyceline-3-3 phosphate dehydrogenase and so on.This experiment measures the activity of lactic acid dehydrogenase, under certain conditions, to a solution containing acetone acid and NADH, add a certain amount of lactic acid dehydrogenase extract, observe NADH in the reaction process 340 nm light absorption reduction value, the more reduced, the higher the LDH vitality. The unit of vitality is defined as 1 enzyme with a drop value of 1.0 per minute at 25 degrees C and pH7.5.reagents
equipmentone. Reagent
50mmol/L pH6.5 hydrophosphate dipotassium-phosphate dihydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrohydrate buffer master: A: 50 mmol/L K2HPO4: K2HPO41.74g plus distilled water dissolved and determined to 200mL..B: 50 mmol/LK2HPO4: KH2PO43.40g plus distilled water dissolved and fixed to 500mL.the solution A 31.5mL and solution B 68.5mL, adjust pH to 6.5. Put a refrigerator in reserve at 4 degrees C.10mmol/L pH 6.5 hydrophosphate di potassium-phosphate potassium dihydrohydrogen buffer was diluted with the above-mentioned mother solution. Ready.0.2 mol/L pH7.5 phosphate di sodium-phosphate dihydrohydrogen sodium buffer master: A: 0.2 mol/L Na2HPO4: Na 2HPO4.12H2O 71.64g plus distilled water dissolved to 1000mL. . B: 0.2 mol/L NaH2PO 4: NaH2PO4.2H2O 31.21g plus distilled water dissolved and settled to 1000mL. the solution A 84 mL and solution B 16 mL, adjust pH to 7.5. Put a refrigerator in reserve at 4 degrees C. 0.1mol/L pH 7.5 phosphate buffer, diluted with the above-mentioned mother solution. Ready. . NADH solution: called 3.5 mg pure NADH
test tube
, plus 0.1mol/L pH7.5 phosphoric acid buffer 1mL shake well. Ready. acetone acid solution: called 2.5mg sodium acetoneate, plus 0.1mol/L pH7.5 phosphoric acid buffer 29mL, so that it is completely dissolved. Ready. two. Material rabbit meat. .