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Application of an affinity electrophoretic and in situ oxidation method to the study of the equine protease inhibitory proteins
Author(s) -
Patterson Scott D.,
Bell Kevin
Publication year - 1989
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150100110
Subject(s) - protease , proteases , chemistry , reagent , isoelectric point , chromatography , biochemistry , electrophoresis , isoelectric focusing , gel electrophoresis , polyacrylamide gel electrophoresis , ligand (biochemistry) , protease inhibitor (pharmacology) , enzyme , biology , receptor , virus , organic chemistry , virology , antiretroviral therapy , viral load
An affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two‐dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second‐dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent ligand in the complex. An in situ oxidation method was developed using the reagent chloramine T to investigate the effect of this reagent on the complexing abilities and inhibitory activities of the protease inhibitory proteins. Oxidation was performed either after electrophoresis prior to staining for enzyme inhibition or during two‐dimensional electrophoresis prior to the aforementioned protease incubation. The latter allowed the effect of oxidation on complex formation to be examined. Whole plasmas were utilized as the sources of protease inhibitory proteins with the human and mouse being used as models. The equine protease inhibitory system was examined by the two methods and shown to consist of three classes of inhibitory proteins based on their susceptibilities to oxidation and abilities to form complexes with various proteases.