Effect of polyethylene glycols on the function and structure of thiol proteases.
Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.
Fatima
Sadaf
S
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202 002, India.
Khan
Rizwan Hasan
RH
D020106:Acrylamide / Q000737:chemistry / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001963:Bromelains / Q000737:chemistry / Q000378:metabolism; D002917:Chymopapain / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008970:Molecular Weight; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature
C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D020106:Acrylamide; D011092:Polyethylene Glycols; D001963:Bromelains; D003546:Cysteine Endopeptidases; D010206:Papain; D002917:Chymopapain