Salt-assisted religation of proteolyzed Glutathione-S-transferase follows Hofmeister series.
Proteases have been used not only for proteolysis but also in organic solvent-assisted religation processes. Here, we demonstrated the effect of salts on peptide bond resynthesis in Glutathione-S-transferase (GST) and have found it to be in the purview of the Hofmeister phenomena. Our results show that the efficiency and ease of religation increases with an increase in the surface charge densities of the cations used in the study. Thus, the yield of religated GST follows the order: Mg2(+)>Li(+)>Na(+)>K(+). Characteristics of the salt-religated GST were studied using size exclusion chromatography, CD spectroscopy, mass spectrometry and CDNB activity assay. Results show that the properties of salt-religated GST are in close agreement with those of the native GST. Additionally, we also assessed the specific activity of the protease, Subtilisin Carlsberg, used in this study. Contrary, to aqueous-organic systems, wherein there is a remarkable decrease in the proteolytic activity, the activity in the presence of salts is only minimally changed. Our studies suggest that salt-assisted peptide bond formation is favoured primarily due to changes in the ionic environment of the nicked termini of GST, and that there is no role played by the protease.
Kishan
K V Radha
KV
Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036 India. radhakishan@gvkbio.com
Sharma
Amit
A
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012492:Salts; D012549:Schistosoma japonicum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000378:metabolism
D010446:Peptide Fragments; D012492:Salts; D005982:Glutathione Transferase; D013381:Subtilisins