Impact of Protein Concentration on the Determination of Thiolic Groups of Ovalbumin: A Size Exclusion Chromatography-Chemical Vapor Generation-Atomic Fluorescence Spectrometry Study via Mercury Labeling

We optimized a hyphenated system based on size exclusion chromatography coupled to a microwave/UV mercury oxidation system and an atomic fluorescence detector (SEC-CVG-AFS) for the online oxidation of free and protein-complexed p-hydroxymercuribenzoic acid (pHMB) without the employment of chemical oxidizing agents. This system has been applied to the study of labeling of thiolic groups of native ovalbumin (OVA) as a function of protein concentration. We found that the protein concentration strongly affects the species distribution of OVA, the number of thiolic groups titrated in each species, and thus, the accuracy in the determination of the total number of thiolic groups. The amount of titrated sulfhydryl groups in the protein concentration range investigated (5-100 mu mol L-1) varied from 2.40 +/- 0.01 to 1.85 +/- 0.05 for the monomeric form of OVA and from 4.63 +/- 0.01 to 5.63 +/- 0.05 for the total OVA, which represents more than four theoretical number of reduced Cys. This information is important from the analytical point of view because it suggests that, unless to operate with diluted concentration of protein, the number of titrated thiolic groups results from both the aspecific interaction of the probe with aggregates species and to the specific bond of the probe with the accessible -SH groups.