PURIFICATION AND PROPERTIES OF A DPNH-TPNH DIAPHORASE FROM CLOSTRIDIUM KLUYVERII

A diaphorase which utilizes both reduced diphosphopyridine nucleotide (DPNH) and reduced triphosphopyridine nucleotide (TPNH) has been purified from a commercial preparation of Clostridium kluyverii. The purified enzyme appears to be homogeneous, and contains flavin mononucleotide (FMN) as a cofactor. The molecular weight of the enzyme is 24,000 and 1 mole of FMN per mole of enzyme is present. No evidence has been found for any metal ion involvement. The enzyme does not catalyze hydrogen exchange between pyridine nucleotides, and cannot utilize DPNH or TPNH to reduce oxygen or cytochrome c. The enzyme is protected against denaturation in urea, guanidine hydrochloride and at elevated temperatures by DPNH, TPNH and FMN, but not by DPN, TPN, FAD or riboflavin. © 1969.