Determination of thiol concentrations in haemolysate by resonance Raman spectrometry

Abstract

A simple and sensitive method for determining the thiol concentration in haemolysate, using the resonance Raman spectrum of the product of the reaction of the lysate with 5,5′-dithiobis(2-nitrobenzoic acid)(Ellman's reagent), is described. The method uses a signal due to the haem ring of haemoglobin as an internal calibrant. In six separate determinations of the thiol concentrations in the lysate from a normal male volunteer, the average thiol concentration was 1 820 µM and the relative standard deviation was 4.5%. Standard addition of glutathione to four lysate samples confirmed this result and indicated an acceptable precision. To obtain a comparison with another technique, clear lysates were prepared by precipitation of haemoglobin and addition of sufficient glutathione to maintain thiol levels comparable to those of coloured lysates. Agreement between the resonance Raman method and a spectrophotometric method was acceptable in this instance. The method is relatively free from the standard interferences found for spectrophotometric assays and is selective. The mean value for the lysate from six normal male volunteers (1920 ± 88 µM) and from seven patients with rheumatoid arthritis (3240 ± 774 µM) were significantly different, suggesting that this measurement may have relevance in the study of disease processes.