Scientia Sinica

No. 1, WANG, TSOU, & WANG: STUDIES ON SUCCINIC DEHYDROGENASE I 89

removed by repeated adsorption on calcium phosphate gel, or by further fractionation with ammonium sulphate at different pH values. It is interesting to note that partial inactivation of the enzyme by exposure to air did not significantly alter the electrophoretic pattern. .

Using the phenazine methosulphate method of assay’ we find that our best preparation has a Qo, of 31000 at 38°C which is approximately twice the activity of Singer, Kearney and Zastrow’s best perparation. Owing to the rapidity with which the purified enzyme loses its activity in the presence of oxygen, it is quite probable that our estimated value does not represent the actual activity attained, In its reaction towards various acceptors, especially phenazine methosulphate, ferricyanide, and methylene blue, our preparation shows general agreement with that of Singer and Kearney!, except for a somewhat lower activity of their preparation with ferricyanide which may be due to the use of a suboptimal concentration of the acceptor in their assay system. According to Kearney, Singer and Zastrow”!, a purified sample of the enzyme prepared in non-phosphate medium showed a specific requirement for inorganic phosphate. At its saturation requirement’ which occurred at or above 0.05 M of phosphate, the enzyme activity was many times higher than that at or below 0,005 M of phosphate. Our purified preparation also exhibits optimal activity at or above a phosphate concentration of 0.05 M, but in disagreement with the observations of these workers!) it shows no spectacular drop of activity when the assay is carried out in borate or imidazole buffer

without the addition of phosphate (a direct determination of phosphate in’

the test system gave a phosphate concentration of less than 3.8 x 10° M). The cause of this discrepancy is still unknown.

The results of our investigations have established the metalloflavoprotein nature of the enzyme specifically concerned with the activation of succinate. The metal involved is non-haematin iron which can be liberated from the protein by denaturation. The flavin prosthetic group is very firmly bound to the apoenzyme and cannot be dissociated from the latter by ordinary treatments, including heat denaturation in weak acid solutions. On boiling the enzyme in dilute acid solution, the iron is split off while the flavin prosthetic group remains attached to the precipitate of denatured protein imparting to it a lemon-yellow colouration. In this property the enzyme differs from the other known flavin-containing enzymes.

The chemical constitution of the flavin prosthetic group as well as the mode of function of the enzyme in the dehydrogenation of succinate is under investigation.

SUMMARY

1. Succinic dehydrogenase has been extensively purified from an aqueous n-butanol extract of modified Keilin-Hartree heart muscle preparation by a