Isolation by gel-permeation chromatography of a non-covalent complex of Cibacron Blue F3G-A with human serum albumin

被引:19
作者
Compagnini, A [1 ]
Fisichella, S [1 ]
Foti, S [1 ]
Maccarrone, G [1 ]
Saletti, R [1 ]
机构
[1] UNIV CATANIA,DIPARTIMENTO SCI CHIM,I-95125 CATANIA,ITALY
关键词
Cibacron Blue F36-A; albumin; proteins; dyes;
D O I
10.1016/0021-9673(95)01362-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The isolation by gel-permeation chromatography on Sephadex G-100 of a non-covalent complex of Cibacron Blue F3G-A (CB) with human serum albumin (HSA) is described. The complex presents a molar ratio of 3:1 CB-HSA and can be re-chromatographed under the same conditions without modification of its composition. However, complete dissociation occurs when the complex is chromatographed in the presence of denaturing agents. The effect of pH on the molar composition of the complex was also investigated by gel-permeation chromatography. Analogous complexes between CB and A and C cyanogen bromide fragments of unreduced HSA were also isolated by gel-permeation chromatography on Sephadex G-50. They present 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 corresponding to subdomain IIA, with the other molecule in the hydrophobic site corresponding to subdomain IIIA. The UV-Vis and dichroic circular spectra of the isolated complexes are reported.
引用
收藏
页码:115 / 123
页数:9
相关论文
共 10 条
[1]  
COMPAGNINI A, 1993, J CHROMATOGR, V639, P34
[2]   SPECTROSCOPIC STUDIES OF CIBACRON BLUE AND CONGO RED BOUND TO DEHYDROGENASES AND KINASES - EVALUATION OF DYES AS PROBES OF THE DINUCLEOTIDE FOLD [J].
EDWARDS, RA ;
WOODY, RW .
BIOCHEMISTRY, 1979, 18 (23) :5197-5204
[3]  
Foster JF, 1977, Albumin: Structure, Function and Uses, P53, DOI DOI 10.1016/B978-0-08-019603-9.50010-7
[4]   ATOMIC-STRUCTURE AND CHEMISTRY OF HUMAN SERUM-ALBUMIN [J].
HE, XM ;
CARTER, DC .
NATURE, 1992, 358 (6383) :209-215
[5]   PROTEIN LIGAND INTERACTIONS STUDIED ON BOVINE SERUM-ALBUMIN WITH FREE AND POLYMER-BOUND CIBACRON BLUE F3G-A AS LIGAND WITH REFERENCE TO AFFINITY PARTITIONING [J].
JOHANSSON, G ;
JOELSSON, M .
JOURNAL OF CHROMATOGRAPHY, 1991, 537 (1-2) :219-233
[6]  
MCLOUGHLIN SB, 1988, REV PROG COLORATION, V18, P16, DOI DOI 10.1111/J.1478-4408.1988.TB00063.X
[7]  
MCMENAMY RH, 1971, J BIOL CHEM, V246, P4744
[8]   SERUM-ALBUMIN [J].
PETERS, T .
ADVANCES IN PROTEIN CHEMISTRY, 1985, 37 :161-245
[9]   DYE-LIGAND AFFINITY-CHROMATOGRAPHY - THE INTERACTION OF CIBACRON BLUE F3GA WITH PROTEINS AND ENZYMES [J].
SUBRAMANIAN, S .
CRC CRITICAL REVIEWS IN BIOCHEMISTRY, 1984, 16 (02) :169-205
[10]   SPECTRAL CHANGES INDUCED IN CIBACRON BLUE F3GA BY SALTS, ORGANIC-SOLVENTS, AND POLYPEPTIDES - IMPLICATIONS FOR BLUE-DYE INTERACTION WITH PROTEINS [J].
SUBRAMANIAN, S .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1982, 216 (01) :116-125