1. The optimum concentration of the substrate; namely, 2ml. of 0.1M succinate were included in 10ml. of the reaction mixture.
2. The optimum concentration of KCN for the inhibition of cytochrome system were found 2-5mM; namely 0.3ml, of 0.1M KCN per 10ml. of the reaction mixture.
3. The optimum pH was found 6.7±0.1 for various tissues of several kinds of fish. A 0.1M phosphate buffer mixture of KH2PO4 and Na2HPO4 (7:3) were employed. The concentration of phosphate buffer of the reaction system was appropriate at 0.1 to 0.2 Mol. When 5ml. of the above buffer solution were included in 10ml. reaction mixture, the resultant pH was usually found 6.6-6.8.
4. The activity of the enzyme increased with rise of reaction temperature up to 50-55°C. Since Q10 above 40°C was higher than at lower temperature, the determination of the activity could be more accurately determined in a lower temperature range. Taking into account the effect due to the temperature difference between the laboratory and the reaction mixture, it was finally decided to carry out the determination at 20°C.
5. The enzymatic activity of homogenized tissue suspension was observed to decrease with time of standing. However, the decrease within a 10 minutes interval was very slight.