A Spectrophotometric Assay for α-Ketoaldehydes Using Horse Liver Alcohol Dehydrogenase

✍ Scribed by C.F. Yang; E.J. Brush

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 384 KB
• Volume: 214
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1465

✦ Synopsis

(\alpha)-Ketoaldehydes have been extensively employed as reagents for the chemical modification of arginine residues in proteins, and to probe for putative anion recognition sites. A major disadvantage in their use is instability, as (\alpha)-ketoaldehydes are prone to hydration, oxidation, and polymerization. These reagents are typically supplied as the aldehyde hydrates which are relatively stable, but must be purified and standardized prior to analytical use. The known chemical, spectroscopic, and enzymatic methods for quantitating (\alpha)-ketoaldehydes are not practical for routine analysis due principally to their high detection limits and sensitivity to interfering substances. Surprisingly, (\alpha)-ketoaldehydes have not been reported as substrates for the alcohol dehydrogenases. We have discovered that phenylglyoxal and several related (\alpha)-ketoaldehydes are good substrates for horse liver alcohol dehydrogenase (HLADH). The second order rate constants (\left(k_{\mathrm{cat}} / \boldsymbol{K}_{m}\right)) are within a factor of 10 of that for the reduction of acetaldehyde, a known good substrate for HLADH. The enzymatic reduction reaction is stoichiometric with the oxidation of NADH, resulting in a rapid, convenient, and sensitive method for the spectrophotometric quantitation of (\alpha)-ketoaldehydes, with a submicromolar detection limit for phenylglyoxal. The sole product of phenylglyoxal reduction has been identified as (\alpha)-hydroxyacetophenone. The ketone functional group is not reduced, and the enzymatic reaction is essentially irreversible as (\alpha)-hydroxyacetophenone is not oxidized to phenylglyoxal by HLADH in the presence of (\mathrm{NAD}^{+}). (c) 1993 Academic Press, Inc.