ABSTRACT
The molecular weight of SOX purified from the cellfree extract is 45,000 daltons. The prosthetic group is a covalently bound FAD. The absorption spectrum of SOX purified from Streptomyces sp. H-7775 has a typical flavoprotein spectrum with the absorption maxima at 276, 358, and 455 nm and a shoulder at 480 nm (Fig. 2A,B). A hypsochromic shift of the second absorption band to 358 nm relative to that of riboflavin at 372 nm has also been observed. The spectrum is similar to those of flavoproteins with covalently bound flavin (11-14). With the addition of D-sorbitol, the peaks at 358 nm and 455 nm decrease owing to the reduction of flavin (Fig. 2C). This indicates that the flavin component is functionally involved in the oxidation of D-sorbitol. The fluorescence intensity of the purified SOX is pH dependent and is similar to that of FAD as in the case for choline oxidase, but different from that of FMN and riboflavin (14). The flavin prosthetic group could not be liberated from the purified SOX protein by (a) acidification with 5% trichloroacetic acid, (b) boiling for 5 min, (c) treatment with 1% SDS, or (d) dialysis against 3 M KBr in 1 mM EDTA for 2 days at 4C (15). It was calculated that 0.9 mol of FAD is bound to 1 mol of SOX, assuming the molecular absorption coefficient for FAD at 460 nm to be 11,300M1cm1 (16).
