Effects of inhibitors on enzyme vitality - the determination of inhibitors and the determination of inhibitor constants (Ki).

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 19test 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 terra-photonometers

"result processing"

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.