Purification and Some Properties of Formate Dehydrogenase

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Abstract
Purified formate dehydrogenase was prepared from dry seeds of pea (Pisum sativum). The enzyme preparation was more than 95% homogeneous in terms of acrylamide-gel disc electrophoresis and the molecular weight was found to be 70,000±2,000 by gel filtration and 72,000±2,000 by sedimentation equilibrium. The pH-activity curve, spectral properties, and amino acid composition were investigated. The enzyme was colorless and contained no significant amounts of iron, manganese, and zinc. Four moles of PCMB (p-chloromercuribenzoate) reacted with the enzyme and the activity of the enzyme was almost completely inhibited as a result. The reaction with PCMB was retarded in the presence of NAD+. On the other hand, DTNB (dithio-bis-nitrobenzoate) was found to react with sulfhydryl groups of the enzyme much faster in the presence of NAD+. Approximately 3.5 moles of sulfhydryl groups per enzyme reacted with DTNB in the presence of NAD+ but the modified enzyme still retained 70% of its original activity. The addition of sodium dodecylsulfate caused further reaction of the enzyme with DTNB and 8 moles of sulfhydryl groups per enzyme were detected in total by the DTNB method.

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