HUMAN 17-BETA-HYDROXYSTEROID DEHYDROGENASE - OPTICAL-PROPERTIES OF ITS COMPLEX WITH NADP(+)

The apoenzyme of the human placental 17 beta-hydroxysteroid dehydrogen ase (17 beta-HSD) and its complex with NADP(+) were prepared from two alternative procedures. The apoenzyme (Form I) has an absorption maxim um at about 279 nm, and an absorption ratio at 280 and 260 nm of 1.65 +/- 0.1; whereas the complex (Form II) has a broad absorption peak bet ween 268-278 nm, and a 280 to 260 nm ratio of 1.1 +/- 0.05. Upon addit ion of the substrate estradiol to the complex, an absorption increase at 340 nm and a fluorescence emission at 450 nm, following NADPH forma tion, were produced. Both changes indicate that one cofactor is tightl y bound to the 17 beta-HSD molecule in this complex. No significant op tical change can be produced in this way for the apoenzyme. Convenient analyses of cofactor content of the enzyme are thus provided. The opt ical analyses and the homogeneous apo- or hole-enzyme preparations are important in the study of the enzyme's function and crystallization. This is the first human steroid converting enzyme which has yielded X- ray quality crystals.