Fluorometric assays for isozymes of human alcohol dehydrogenase.
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.
Wierzchowski
J
J
Center for Biochemical and Biophysical Sciences, Harvard Medical School, Boston, Massachusetts 02115.
Dafeldecker
W P
WP
Holmquist
B
B
Vallee
B L
BL
D000426:Alcohol Dehydrogenase / Q000032:analysis*; D000447:Aldehydes / Q000032:analysis; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology; D009281:Naphthalenes / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000447:Aldehydes; D005456:Fluorescent Dyes; D007527:Isoenzymes; D009281:Naphthalenes; C059869:4-methoxy-1-naphthalaldehyde; C059868:6-methoxy-2-naphthalaldehyde; D000426:Alcohol Dehydrogenase