Absolute on-line molecular mass analysis of basic fibroblast growth factor and its multimers by reversed-phase liquid chromatography with multi-angle laser light scattering detection.

The multi-angle laser light scattering (MALLS) detection method was combined with reversed-phase high-performance liquid chromatography to analyze multimerization of basic fibroblast growth factor (bFGF) formed by oxidation of bFGF with air or with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). This analysis provided the absolute molecular mass and the mean square radius for each eluted protein fraction or each slice of the chromatogram. It was shown that depending on the oxidation conditions, bFGF forms different multimeric forms, from dimers to hexamers. It was found that these multimers have varied conformations of the same molecular mass, but different structure. Molecular mass and size analyses provided molecular conformation of the aggregates: the results indicated the formation of rod-like rigid structures. The MALLS analysis confirmed that, during oxidation, each bFGF monomer bound sequentially to form the extended multimer. The proposed scheme of bFGF oxidation with DTNB revealed that the difference in the aggregate structural forms was probably due either to the presence of covalently bound residues of nitrobenzoic acid in the products of oxidation, or to the participation of sulfhydryl groups in disulfide bond formation.

Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations.

A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p beta-Gal plasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium+ ++) bromides random copolymer (C16PVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma "Jurkat", and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The in vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of beta-galactosidase (beta-Gal) expression using 4-methylumbeliferril beta-D-galactopyranoside as a substrate (p beta-Gal plasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)