Wierzchowski J, Dafeldecker W P, Holmquist B, Vallee B L
Center for Biochemical and Biophysical Sciences, Harvard Medical School, Boston, Massachusetts 02115.
Anal Biochem. 1989 Apr;178(1):57-62. doi: 10.1016/0003-2697(89)90356-4.
Two new fluorogenic substrates for human alcohol dehydrogenase (ADH), 4-methoxy-1-naphthaldehyde (IA) and 6-methoxy-2-napthaldehyde (IIA), are described. The 2-naphthaldehyde derivative fluoresces in aqueous media with a quantum yield of 0.22 with an emission maximum at 450 nm, but the 1-naphthaldehyde shows only weak fluorescence. The corresponding alcohol reduction products, 4-methoxy-1-naphthalenemethanol (IB) and 6-methoxy-2-naphthalenemethanol (IIB), exhibit fluorescence in the near uv region with quantum yields of 0.36 and 0.26, respectively. The Km values for the individual homogenous class I ADH isozymes, with the above naphthaldehydes as substrates, range from 0.35 to 11.5 microM. The kappa cat values range from 70 to 610 min-1 and are thus comparable to those for the best ADH substrates. Except for the beta 1 beta 1 isozyme, IA is the preferred substrate for class I ADH isozymes while IIA is rapidly reduced by class II (pi-ADH). The sensitivity and specificity of the enzymatic assay with IA as substrate are demonstrated and provide the basis for the determination of class I ADH activity in human serum.
本文描述了两种用于人类乙醇脱氢酶（ADH）的新型荧光底物，4-甲氧基-1-萘甲醛（IA）和6-甲氧基-2-萘甲醛（IIA）。2-萘甲醛衍生物在水性介质中发出荧光，量子产率为0.22，最大发射波长为450nm，但1-萘甲醛仅显示出微弱的荧光。相应的醇还原产物，4-甲氧基-1-萘甲醇（IB）和6-甲氧基-2-萘甲醇（IIB），分别在近紫外区域发出荧光，量子产率分别为0.36和0.26。以上述萘甲醛为底物时，各个I类ADH同工酶的Km值范围为0.35至11.5μM。催化常数（kappa cat）值范围为70至610分钟-1，因此与最佳ADH底物的值相当。除β1β1同工酶外，IA是I类ADH同工酶的首选底物，而IIA则被II类（π-ADH）迅速还原。以IA为底物的酶促测定的灵敏度和特异性得到了证明，并为测定人血清中I类ADH活性提供了依据。
