Comparative fluorescence studies of native and crosslinked glucose oxidase

✍ Scribed by Beca Solomon; Meir Shinitzky

• Publisher: Wiley (John Wiley & Sons)
• Year: 1977
• Tongue: English
• Weight: 444 KB
• Volume: 16
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.1977.360160903

✦ Synopsis

Abstract

The binding characteristics of flavin adenine dinucleotide (FAD) to apoenzyme preparations obtained from native and intramolecularly crosslinked glucose oxidase were determined and compared. The dissociation constants K~diss~ as well as rates of recombination of FAD with the two apoenzyme preparations, were independently evaluated from fluorescence quenching of either the tryptophans of FAD. The K~diss~ values thus obtained were <10^−19^M for native glucose oxidase and 4 ± 1 × 10^−7^M for the crosslinked enzyme. The recombination of apo glucose oxidase with FAD, which is presumably diffusion controlled, is followed by an apparent first order decrease in fluorescence intensity of both the protein tryptophans and FAD, with a rate constant around 0.2 min^−1^. This could be related to conformational changes which occur immediately after binding of FAD to the apoenzyme, an interpretation which is supported by the markedly different results obtained in the analogous experiments with the crosslinked enzyme. A model for the conformational characteristics of glucose oxidase, based on this study, is proposed.