Scientia Sinica

No. 1 WANG, TSOU, & WANG: STUDIES ON SUCCINIC DEHYDROGENASE I 87

2 ss Olt g 2 c Vv 3 es vu oO a vo 3 a a Oo mi Ss Vv eS —— —— 1 1 \ i == 7 2 5 4 5 6 7 pH

Fig, 9. The effect of pH on the fluorescence intensity of the prosthetic group. Relative fluorescence intensity is plotted’ against pH. The sample used was a proteolytic enzymes-digested-dehydrogense preparation.

The iron content was determined after acid digestion by the , a’ -dipyridy! method and the flavin content either by the decrease in absorption at 450 mu after Na-dithionite reduction or from the intensity of fluorescence at pH 3 after being treated with proteolytic enzymes and calculated on the assumption that the intensity is the same as flavin-adenine dinucleotide?™. It was found that the highly purified enzyme contained 1 gram-mole of flavin ané 4 gram-atoms of iron for every 140,000-160,000 grams of the enzyme protein. Occasionally, lower iron to flavin ratio was found in certain preparations of lower specific activity. Both the iron and the flavin components are tightly bound tothe protein part of the enzyme and cannot be removed by prolonged dialysis or repeated precipitation of the enzyme with ammonium sulphate. When the enzyme solution has been digested by treatment with proteolytic enzymes, the absorption spectrum of the solution is also changed, so that it no longer shows an absorption maximum at 415 mp, and the 460 mp shoulder is shifted to 450 mp. All these seem to indicate that both iron and flavin are integral parts of succinic dehydrogenase. It also appears probable that the 415 mu maximum is due to the linkage between the prosthetic group and the protein.

Separation of the flavin prosthetic group. After proteolytic digestion, the enzyme solution was acidified and made 0.7 saturated with ammonium sulphate by the addition of the solid salt. The prosthetic group* was then extracted into p-cresol and thoroughly washed by acid ammonium sulphate solution. It was again transferred into aqueous solution by adding ether to the p-cresol solution, precipitated as the mercury salt and the latter decomposed

*In what follows the word “prosthetic group”. refers to the product of proteolytic action which contained the flavin prosthetic group of the enzyme.