Table 1. Mechanisms of chemical inactivation of proteins and experimental methods for their study
| Mechanism of inactivation | Methods for identifying changes in protein structure |
|---|---|
| Aggregation (sometimes followed by formation of intermolecular S-S bonds) | Formation of visible precipitates Detection of aggregates by native electrophoresis, size exclusion chromatography, or sedimentationDissociation of aggregates by denaturants (e.g., 6 M guanidine HCl, 8 M urea); if cross-linked with S-S bonds, thiols are added. |
| Changes in primary amino acid: Hydrolysis of peptide bonds by mild acid conditions (fastest on the carboxyl side of Asp residues, especially Asp-Pro bonds)Oxidation of Cys, Trp, and MetReduction of S-S bonds by OH− followed by β-elimination and reactions of formed SH groups either with S-S bonds of cysteine or with other amino acidsModification of SH groups by mixtures of metal ions to form mercaptidesDeamidation of Asn and GlnRacemization of amino acids |
Low-molecular-weight protein fragments are displayed by SDS-PAGE under reducing conditions. Retardation of protein inactivation under inert gases (e.g., nitrogen)Detection of emerging SH groups by Ellman’s reagent (Ellman 1951)Inhibition of disulfide exchange by reagents that block SH groups (e.g., alkylating reagents such as iodoacetic acid and iodoacetamide) and acceleration of the reaction with thiolsProtein reactivation by breaking “non-native” S-S bonds with thiolsDisappearance of Cys, Lys, and Arg and emergence of lysinoalanine, dehydroalanine, and ornithino-alanine detected by amino acid analysisRetardation of protein inactivation by EDTADecrease in free SH groups is detected by Ellman’s reagent.Detection of new protein forms with lower isoelectric points by isoelectric focusing; specific sites of deamidation can be determined using a combination of peptide mapping and mass spectrometry.Contribution from the process, particularly aspartic acid and serine racemization, is feasible only during long incubation at elevated temperatures; amino acid racemization can be detected by amino acid analysis (Bada 1984). |
| Elimination of coenzyme from the active site | Disappearance of characteristic absorption bands in protein spectra |
| Dissociation of oligomeric proteins into monomers | Dissociation products are detected by size-exclusion chromatography or non-denaturing electrophoresis. |
| Adsorption on the surface of the vessel | Elimination of inactivation by preliminary adsorption of an inert protein. Desorption is sometimes accomplished by denaturants. |
| “Incorrect refolding” with production of mismatched protein forms | Retardation of thermoinactivation by denaturants. Protein unfolding is monitored by changes in fluorescence, UV absorbance,
circular dichroism (CD) (Pace and Scholtz 1997; Schmid 1997), gel electrophoresis (Goldenberg 1997), and differential scanning
calorimetry (DSC) (Gilleto and Pace 1996). Reactivation of mismatched forms by unfolding with reduction of S-S bonds and subsequent refolding with reoxidation of S-S bonds (Mozhaev et al. 1987) |
| (Adapted, in part, from Mozhaev 1993 with permission from Elsevier © 1993.) | |
