Purification and characterization of commercial NADH and accompanying dehydrogenase inhibitors

✍ Scribed by I. Wenz; W. Loesche; U. Till; H. Petermann; A. Horn

• Publisher: Elsevier Science
• Year: 1976
• Tongue: English
• Weight: 656 KB
• Volume: 120
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(01)99011-9

✦ Synopsis

The anion-exchange chromatography of commercial NADH using a potassium bicarbonate solution as eluent yields highly pure NADH with good stability_ Twelve compounds are also separated which act as dehydrogenase inhibitors. The main impurities are further characterized. The compound mainly responsibie for residual optical density in commercial NADH preparations is probably a stereoisomer of NADH which is in reversible equilibrium with NADH at pH values in the range 5-7. A method of thin-layer chromatography, to check commercial NADH preparations for impurities, is described_

INlRODUCTION

Accurate measurements of the activities of enzymes requiring NADH as coenzyme depend largely on the purity of the NADH. The formation of inhibitory compounds in NADH preparations was originally reported by Lowry et ~1.~ and Dalziel' and subsequently confirmed by many other authors. A review on this subject by Gerhardt eb al.' has recently appeared_ Difficulties are encountered in purifying the coenzyme and assuring its stability. The unfavourabie influence of humidity, freezing or acidification on its stability is well. established. In previous papers we reported a new chromatographic procedure with potassium bicarbonate solution as an easily removable eluent3p4. The present paper deals with the application of this method to the purification of commercial NADH which results in a highly purified product with I excellent stability. More than ten compounds are removed during the purification procedure, all of which inhibit several NADH-dependent dehydrogenases. Possible structures for the main impurities are discussed_ Conditions leading to their formation from NADH were investigated.