Takeyoshi Imamura

High-Speed Gel Filtration of Polypeptides in Some Denaturants

Abstract The separation and analysis of proteins and polypeptides by use of a silica-based gel packing, G3000SW, for high-speed gel filtration are investigated. The peaks of bovine serum albumin, pepsin, trypsinogen, myoglobin and cytochrome c were completely separated in the presence of 0.2% SDS and 0.2 M sodium phosphate buffer (pH 7.0). The elution positions of native proteins, polypeptides in 8 M urea and polypeptide-SDS complexes were influenced by the concentration of sodium phosphate in eluents, though those of polypeptides in 6 M guanidine hydrochloride were little. These facts suggest the presence of the electrostatic interactions between negatively charged gel surfaces of the packing and polypeptides. Taking into account of the interactions, it is shown that the high-speed gel filtration by use of this column is available to the rapid estimation of molecular weight of polypeptides in both systems of SDS and 6 M guanidine hydrochloride.

Interaction of indole and tryptophan derivatives with sodium dodecyl sulfate micelles measured with ultraviolet absorption and fluorescence quenching

The distribution of indole and tryptophan derivatives between sodium dodecyl sulfate (SDS) micellar and aqueous phases was analyzed using conventional methods of ultraviolet (UV) absorption spectroscopy and measurement of fluorescence quenching by succinimide. On the assumption of a simple pseudo-phase equilibrium between both phases the distribution coefficient was easily obtained by the measurement of the ratioRpv of the absorbance intensity in the peak to that in the valley of the UV spectra or the fluorescence quenching constant Ksv. The possibilities and limitations of utilizing the ratio of the collisional quenching constant estimating from theKsv value in the micellar phase to that in the aqueous phase for a measure of the polarity of the microenvironment around the tryptophan derivatives in the SDS micelle is discussed in comparison with theRpv values for the UV spectra. The indole ring in the derivatives in the SDS micelle is localized near or on the micelle-water interface with its imino group directed toward the aqueous phase. Thus it can serve as a feasible model for interpreting the distribution coefficients andRpv values obtained for the various indole and tryptophan derivatives.

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS)n.

Microenvironment of tryptophan residues in β-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes

The changes of microenvironment of tryptophan residues in β-lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) β-lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM β-lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.