THE EFFECT OF PH ON RAPESEED GLOBULIN (CRUCIFERIN) BINDING TO ANILINONAPHTHALENE-8-SULFONATE

The fluorescence emission intensity from rapeseed globulin (cruciferin ) increased in the presence of anilinonaphthalene-8-sulfonate (ANS) at pH 2.0 but not at pH 10. Fluorescence titration studies showed that a t pH 7 cruciferin binds 22 (+/- 0.6) mobs of ANS per mole of protein w ith an average dissociation constant (K-d) of 1.9 (+/- 0.1) x 10(-5) M . At pH 2.0 the number of ligand binding sites (n) decreased to 14 (+/ - 0.2) moles of ANS bound per mole of cruciferin, However, the ANS bin ding affinity increased by about five times (K-d = 3.6 (+/- 1.1) x 10( -6) M). The fluorescence emission spectrum maxima (lambda(max)) for th e cruciferin-ANS complex showed a blue shift at pH 2 when compared to lambda(max) values at pH 7-10. These results are consistent with a los s of the quatemary and tertiary structures of cruciferin and the expos ure of surface hydrophobic ANS binding sites at low pH. Cruciferin-ANS binding parameters at pH 10 were not significantly different from val ues at pH 7; n = 22 and K-d = 2.7 (+/- 0.2) x 10(-5) M. Based on these ANS fluorescence measurements crucifer n is stable under alkaline con ditions.