ISOLATION BY GEL-PERMEATION CHROMATOGRAPHY OF A NONCOVALENT COMPLEX OF CIBACRON-BLUE F3G-A WITH HUMAN SERUM-ALBUMIN

The isolation by gel-permeation chromatography on Sephadex G-100 of a non-covalent complex of Cibacron Blue F3G-A (CB) with human serum albu min (HSA) is described. The complex presents a molar ratio of 3:1 CB-H SA and can be re-chromatographed under the same conditions without mod ification of its composition. However, complete dissociation occurs wh en the complex is chromatographed in the presence of denaturing agents . The effect of pH on the molar composition of the complex was also in vestigated by gel-permeation chromatography. Analogous complexes betwe en CB and A and C cyanogen bromide fragments of unreduced HSA were als o isolated by gel-permeation chromatography on Sephadex G-50. They pre sent a molar ratio of 0.8:1 and 1.3:1 CB-protein for fragments A and C , respectively. These results suggest that two of the three molecules of CB bound to HSA may be located in the hydrophobic pocket correspond ing to subdomain IIA, with the other molecule in the hydrophobic site corresponding to subdomain IIIA. The UV-Vis and dichroic circular spec tra of the isolated complexes are reported.