ISOLATION AND BIOCHEMICAL-CHARACTERIZATION OF HIGHLY PURIFIED ESCHERICHIA-COLI MOLECULAR CHAPERONE CPN60 (GROEL) BY AFFINITY-CHROMATOGRAPHYAND UREA-INDUCED MONOMERIZATION

Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been f urther purified from tightly bound substrate polypeptides by two diffe rent procedures: (i) group-specific affinity chromatography by using t he triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea -induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the m ajority of proteins, but has no affinity to GroEL because of its low c ontent of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are ex posed on its surface and available for affinity-ligand interactions an d the complex is thereby retarded on Procion yellow columns. Pure subs trate-free GroEL was obtained after ion-exchange chromatography of Gro EL monomers followed by reassembly of the purified monomers into funct ional GroEL oligomers. The final preparation contained no substrate po lypeptides bound to GroEL as judged by electrophoretic analysis and la ck of tryptophan fluorescence. GroEL preparations also displayed two e qually strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.