Preparation of buffer for determination of citrate synthase;

Sample buffer PH 7.5 100

Tris/HCl 50 mM 0.6055 g

KCl 100 mm 0.7455 g

EDTA 1 mm 0.03722g

Storage liquid110ml

Acetyl coenzyme A 2.5 mm 20.225 mg

Store solution 210ml.

Oxoxalacetic acid (OAA)5 mm 6.6035 mg

Storage solution 310ml

DTNB 5.02519.914mg.

Steps: 520 microliters of sample buffer +20 microliters of TNB+20 microliters of acetyl coenzyme a+20 microliters of enzyme solution to be detected, and warm bath at 25℃ for 5 minutes. Immediately after adding 20ulOAA, put it into a spectrophotometer with a constant temperature of 25℃, and measure the change value of optical density at the wavelength of 4 12nm for 3 minutes (record the optical density every 30 seconds). The measurement was repeated at 25℃, and the average value was taken as the measured value, and the unit of enzyme activity was expressed as U/min/g.

Calculation: u/min/g = (Amin-A0)/min/mg (protein).

This calculation formula is not very clear.

This method comes from "adaptive changes and mechanism of myocardial and skeletal muscle in rats with hypoxia-hypoxia combined exercise" (doctoral thesis of the Third Military Medical University, 2005).