Role of protein-solvent interactions in refolding: effects of cosolvent additives on the renaturation of porcine pancreatic elastase at various pHs.
The effects of cosolvent additives on the refolding of porcine pancreatic elastase were studied by comparing the enzymatic activity and the conformation of the enzyme renatured at various pHs with those of the native elastase under the same cosolvent and pH conditions. A lag period was observed before reaching the steady state of the hydrolysis of an amide substrate, and the lag period measured with the refolding enzyme was longer than that measured with the native elastase. Depending on the cosolvent studied (acetonitrile, dimethylsulfoxide, glycerol, methanol) there was or was not a dramatic increase in the duration of the lag period measured with the refolding enzyme, but not in the case of the native elastase. These results and additional kinetic data on inactivation of the enzyme demonstrated that dimethylsulfoxide, glycerol, and methanol enhance the stability of the intermediates able to refold into the native form, contrary to acetonitrile. In neither the case of the native enzyme nor that of the renatured enzyme, did the cosolvents modify the pK(app) of ionization of the amino acids that control enzymatic activity, indicating that they did not penetrate the core of the refolded elastase. Conversely, they shifted toward a more alkaline pH the structural transition of the native elastase, and the amplitude of the shift was comparable to that observed in bulk water with elastase whose Ser 195 has been acylated, suggesting that cosolvents stabilized the structure of the folded molecule by increasing its packing.
Jaspard
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Laboratoire de Physicochimie des Protéines, Université Paris Sud, Paris, France. jaspard@univ-angers.fr
D000097:Acetonitriles / Q000378:metabolism; D000215:Acylation; D000577:Amides / Q000378:metabolism; D000818:Animals; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004795:Enzyme Stability; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D012694:Serine / Q000378:metabolism; D012997:Solvents / Q000378:metabolism*; D013552:Swine
D000097:Acetonitriles; D000577:Amides; D012997:Solvents; D012694:Serine; D010196:Pancreatic Elastase; D005990:Glycerol; D000432:Methanol; D004121:Dimethyl Sulfoxide; C032159:acetonitrile