Scientia Sinica

No. I WANG, TSOU, & WANG: STUDIES ON SUCCINIC DEHYDROGENASE I ; 75

the addition of 2.5 ml of water. The precipitate was removed by centrifugation and the light absorption of the supernatant was read against a blank containing 2 mM K;Fe(CN), in the spectrophotometer (Beckman, Model DU) using cells of 10 mm light path. An enzyme preparation causing a decrease of optical density of 0.100 per mg N at 0°C is defined as having a specific activity of 1. Within the range of concentrations used, potassium ferricyanide obeys Beer’s law and its molecular extinction coefficient at 420 mp is 1.03 x 10°.

In some experiments, the rate of ferricyanide reduction was directly followed in the spectrophotometer. In such cases, the reaction mixture had the same composition except that the reaction was carried out in Beckman cells of a 5 mm light path. The blank is a lcm Beckman cuvette containing 2 mM ferricyanide solution. When constant temperature was desired, the apparatus previously described was employed”.

Using this assay method, the enzyme activity was found to be directly proportional to the amount of enzyme protein present as can be seen in Fig. 1. The reaction follows a linear course for over 10 minutes as is shown in Fig. 2.

0.f 0.2 0.5

ml of enzyme solution

Fig. 1. Relation between enzyme concentration and rate of ferricyanide reduction. The reaction was carried out in 5 mm Beckman cells at 38°C. The decrease in optical density at 420 mp after 24% min. reaction is plotted against ml of a stock enzyme solution added to 3.5 ml of reaction mixture, The stock enzyme solution had a specific activity of 76 and a concentration of 0.683 mg N/ml.

Protein determinations. ‘These were carried out either colorimetrically with the biuret reagent!" or turbidimetrically with trichloroacetic acid!””. Both methods were standardized against nitrogen determination by micro-

Kjeldahl.