Scientia Sinica

No. l WANG, TSOU, & WANG: STUDIES ON SUCCINIC DEHYDROGENASE I : 77

separated by centrifugation at 0-2°C. The precipitate, when dissolved in a small amount of 0.075 M: phosphate buffer, pH 7.2, was amber in colour and had a specific activity of 90-120.

4. Second ammonium sulphate fractionation. The protein content of the solution obtained above was determined and the solution was diluted until the final protein concentration was approximately 0.6%. The pH was adjusted to 7.2 (phenol red) and the ammonium sulphate content was determined by Nessler’s reagent. Neutralized ammonium sulphate solution (0.9 saturation) was slowly added in the cold and the fraction precipitated between 0.4-0.5 saturation of ammonium sulphate was collected and dissolved in a small amount of 0.04 M.phosphate buffer pH 7.8. The final preparation is a clear amber-coloured solution with a specific activity of 200-250.

The main purification steps are summarized in Table 1. Table 2 shows the increase in specific activity and the recovery of enzyme activity at each step of purification.

Table 1. Scheme-of Purification of Succinic Dehydrogenase.

Pig heart muscle mince

Cytochrome c-deficient heart muscle preparation

| Treatment with 0.2 volume:

| n-butanol vv | oe Residue Aqueous solution discarded Adsorption on calcium phosphate v gel | : | Supernatant Gel discarded Phosphate buffer elution V | | Gel Eluate discarded

| Ammonium sulphate fractionation

Fraction precipitated between 0.35-0.55 saturation dissolved in phosphate buffer

Second ammonium sulphate frac| tionation Fraction precipitated between

0.4-0.5 saturation dissolved in phosphate buffer