acid phosphatase kinetic properties analysis
inhibitors on enzyme vitality - the determination of the type of inhibitor and the determination of the inhibitor constant (Ki.
inhibitors can be divided into two types of irreversible inhibition and reversible inhibition according to the characteristics of inhibitor-enzyme binding, among which reversible inhibition can be divided into three types: competitive inhibition, non-competitive inhibition and anti-competitive inhibition.
(1)competitive inhibition type:
enzyme cannot bind to substrates and inhibitors at the same time. Dynamic characteristics are: the surface meter constant Km "increase, Vm" unchanged. (Ki in the lower version is the inhibitor constant, and the concentration of the inhibitor is the inhibitor concentration).
(2) non-competitive inhibition type:
inhibitors, substrates can bind to enzymes at the same time, but this complex cannot be further decomposed into products, Km "unchanged, Vm" decreases.
(3) anticompatitive suppression type:
inhibitors must bind to the enzyme to form a compound with the enzyme, but this material cannot be decomposed into products. Km", Vm" are all changed.
for the enzyme-promoting reaction with inhibitors, it is also available to use 1/v to 1/S" as a graph, to find Out Km", Ki, Vm", and use the dynamic characteristics of various reversible inhibition types to determine the type of inhibition.
takes 19 test tubes0 to 18 numbers, tube 0 is blank. Each tube is added to the table below the table with a different volume of 5mmol/L phenyl phenylphate sodium disodium solution (pH5.6) and shake well. Add l0mmol/L phosphate potassium dihydrohydrogen 0.1mL or 3mmol/L sodium fluoride solution 0.1mL and supplement 0.2mol/L pH.5.6 acetate buffer to each tube solution with a total volume of 0.6mL, shake well, at 35 degrees C constant temperature Each tube timed to add a diluted enzyme solution 0.4mL, fully shake well, 35 degrees C accurate reaction 15min, add 1mol/L sodium carbonate solution 2mL, and then add 0.5mL Folin-phenol dilute solution, continue to insulation color 10min. The 0.4mL enzyme fluid in tube 0 is finally added, and the other operations are the same as in the 6th tube of the inhibitor-free group. After cooling, the tube No. 0 is blank
1, calculated tubes ( S) , 1 / S) values;
2, according to the absorbance A680 of each tube is worth a considerable phenol content / smol (see experiment 31), calculated v, 1/v value;
3, KH2PO4 and NaF inhibitors 1/v to 1 / s Curves (with inhibitors and no inhibitors are made in the same axis), the type of inhibition of KH2PO4 and NaF inhibitors is determined;
4, calculated in the same axis, and the corresponding inhibitor constant Ki is calculated.