Applications of the in vacuo chemical modification technique to the study of protein structure and function.
|Title:||Applications of the in vacuo chemical modification technique to the study of protein structure and function.|
|Authors:||Vakos, Helen T.|
|Abstract:||Sensitive NMR methods have been developed for analyzing N- and C-termini in protein structure for investigating protein purity, identity, and homogeneity. The in vacuo chemical modification procedure allows for the incorporation of isotopically labeled reagents (13C, 14C, 2H, 3H) into protein with a high degree of reaction specificity in a cost-effective manner, since there is no competing reaction with water and only microlitre quantities are required to derivatize milligram quantities of lyophilized protein. Reaction in vacuo at 75°C with 13C-iodomethane results in the preferential 13C-trimethylation of alpha-amino groups in proteins lyophilized from aqueous solutions at pH (LpH) 6 to 7. In vacuo trimethylation of alpha-amino groups and analysis by 13C-NMR spectroscopy provides a new approach for determining the number and types of N-terminal amino acid residues and for verifying the presence of a free or blocked amino-terminus in a protein preparation. Reaction of lyophilized proteins in vacuo at 75°C with 13C-iodomethane at LpH values between 3 and 4 effects the preferential esterification of carboxyl groups, and is in accord with the pH memory effect. The extent of reaction increases with an increase in amount of deprotonated carboxyl groups at higher LpH values, but trimethylation of amino groups is the predominant reaction at LpH values greater than 4 or 5. In contrast, the novel in vacuo esterification reaction (75°C) of lyophilized proteins (LpH 3) with gaseous 13C-methanol/HCl or 13C-ethanol/HCl results in the exclusive, rapid, and extensive formation of alpha-, gamma- and delta-carboxyl 13C-methyl or 13C-ethyl ester derivatives with no protein degradation, which could be distinguished by the distinct chemical shifts of their resonances. Reaction in vacuo with 14C- or 3H-enriched iodomethane results in a higher incorporation of specific radioactivity into lyophilized proteins than can be achieved in aqueous environments. The chemical tag can serve as a probe of structure and function in tracer studies for proteins that are not enzymes. Trace-radiolabeled proteins (e.g., 14C-methylated Bacillus thuringiensis (Bt) insecticidal toxin, 14C-methylated insulin) show a high specific activity, and retain bioactivity (e.g., 14C-methylated Bt toxin). In a separate study, pure bovine and porcine cholesterol esterase (CE) and human milk bile salt-activated lipase are obtained using a cholate-derivatized affinity column and a taurocholate concentration gradient in the eluent, which is critical to the success of the method. Taurocholate induces dimerization of bovine CE in SIDS as observed by SDS-PAGE. Studies of the bile salt-modulated stereoselectivity of CE-catalyzed hydrolysis of alpha-tocopheryl acetates show that, in competitive experiments, the diastereoselectivity of CE depends on the bile salt used, which is relevant if the bile salt-modulated effect on CE-catalyzed reactions is to be exploited in organic syntheses. (Abstract shortened by UMI.)|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|