Scientia Sinica

76 SCIENTIA SINICA Tol. .V

0.50

—Ad 3 &

5 10 Time (min.) Fig. 2, Course of ferricyanide reduction. Conditions as in Fig. 1 except that the reaction was carried out at room temperature (about 32°C), and the reaction mixture contained 0.0076 mg of enzyme N with a specific activity of 156. : . Micro-electrophoresis. This was carried out with the Antweiler micro-

electrophoresis apparatus. -

PurIFICATION

1. Butanol extraction. Yo each litre of cytochrome c-deficient heart muscle preparation suspended in borate buffer of 0.05 M final concentration, pH 8, were added 60 ml of 0.8 M sodium succinate and 100 ml of neutralized 0.1 M KCN. After being cooled to 0°C and allowed to stand at this temperature for 2 hours, the pH of this mixture was slowly adjusted to 9 by the addition of N NaOH (slight pink to a mixture of phenolphthalein and thymolphthalein). Pre-cooled #-butanol, 1/5 the volume of the mixture, was added gradually so that the temperature remained below 15°C. After all the butanol had been added, the mixture was allowed to stand for 30 minutes with occasional stirring. When separated by centrifugation, the middle aqueous layer was lightly yellow in colour and had a specific activity of 15-25.

2. Adsorption on calcium phosphate gel. The cold butanol extract was adjusted to pH 6 (methyl red) with N-acetic acid. Calcium phosphate gel was added to a final concentration of 3.6 mg/ml. The mixture was stirred for ) minutes and centrifuged and the supernatant was discarded. The precipitate was eluated by stirring for 15 minutes with 4/5 of the volume of the extract of cold 0.075 M phosphate buffer, pH 7.8. The eluate obtained by centrifugation was yellow in colour, perfectly clear and had a specific activity of 45-70.

3. First ammonium sulphate fractionation. The eluate was adjusted to pH 7.2 (phenol red) by N-acetic acid and solid (NH.),SO, added. The precipitate formed between 0.35-0.55 saturation of ammonium sulphate was