ALPHA-GLUCAN BINDING OF POTATO-TUBER STARCH-BRANCHING ENZYME-I AS DETERMINED BY TRYPTOPHAN FLUORESCENCE QUENCHING, AFFINITY ELECTROPHORESISAND STEADY-STATE KINETICS

The affinity of potato tuber starch-branching enzyme-I (PSBE-I) for va rious linear malto-oligosaccharides, cyclodextrins, (CDs) and macromol ecular alpha-glucans was investigated by alpha-glucan induced fluoresc ence quenching of intrinsic PSBE-I tryptophan residues and by affinity electrophoresis. alpha-Glucan binding was characterised by distinct s hifts towards shorter wavelengths of the PSBE-I fluorescence emission spectrum and by concomitant reductions in fluorescence intensity. The magnitudes of both the maximum shift in emission spectrum and reductio n in fluorescence intensity were dependent on the alpha-glucan ligands used. Maximum K-d for a range of linear malto-oligosaccharides analys ed was 0.13 mM as found at a degree of polymerisation (DP) of 13. Larg e differences in dissociation constants were measured for CDs with DP 6 (alpha-CD, 6.0 mM), DP 7 (beta-CD, 0.25 mM) and DP 8 (gamma-CD, 0.67 mu M). The high-molecular-mass alpha-glucans amylose and amylopectin, both substrates for PSBE-I, showed apparent affinities of 0.018 and 0 .066 mg/ml, respectively. Small linear and cyclic oligosaccharides com peted with amylopectin in the affinity electrophoresis system and they were also competitive inhibitors for PSBE-I activity. The affinities for oligosaccharides as measured by competition were, however, about 1 0-fold lower than as measured by fluorescence quenching suggesting the existence of a separate oligosaccharide binding site on PSBE-I. Affin ity electrophoresis revealed multiform heterogeneity in the enzyme pre paration with respect to alpha-glucan interaction.