Junko Nishiyama

Measurement of biological thiols and disulfides by high-performance liquid chromatography and electrochemical detection of silver mercaptide formation.

A rapid and sensitive method is described for the measurement of picomole levels of the biological thiols glutathione, cysteine, penicillamine, cysteamine, and ergothioneine by a combination of high-performance liquid chromatography and electrochemical detection (ECD). The compounds were separated isocratically on a reversed-phase C18 column by ion-pair chromatography with a mobile phase containing 5 mM acetic acid and 2.5 mM sodium 1-octanesulfonate. After chromatographic separation, the eluate was combined with silver nitrate dissolved in ammonium nitrate buffer at pH 10.5. A platinum disc electrode was used at -0.1 V vs Ag/AgCl to detect the amount of silver ions that had been consumed by the reaction with thiols. For measurement of disulfide, S-sulfonation with sodium sulfite or electroreduction were used to cleave the disulfide, and the thiol anions produced were detected by HPLC-ECD as for the reduced forms. The method was used to assay thiols and disulfides in biological materials.

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30 degrees C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.

A Spin-Label Study on Lipids in Dough Formed at Various Concentrations of Oxygen

The effect of oxygen on wheat flour lipids during dough mixing was investigated by analysis of the lipid composition and by an ESR technique with a fatty acid spin-label (4,4’-dimethyl-oxazolidine-N-oxyl derivative of 5-ketostearic acid). Dough was prepared in the presence of the spin-label under an atmosphere of air, nitrogen, 95% nitrogen—5% oxygen or oxygen, and the gluten was obtained by washing out the starch. ESR spectra of the spin-label incorporated into the gluten showed decreases in the order parameter, rotational correlation time and activation energy for rotational viscosity with increasing atmospheric oxygen concentration. During dough mixing in oxygen, oxidation of lipids proceeded and bound lipids slightly decreased. These data indicate that modification of lipids by incorporated oxygen leads to an increase in their fluidity and to a decrease in their hydrophobic interaction with protein in dough.