Induction of 'molten globule' like state in acid-denatured state of unmodified preparation of stem bromelain: implications of disulfides in protein folding.
A denatured state of unmodified preparation of stem bromelain representing a structureless form has been characterized at pH 2.0 and the effect of increasing concentration of TFE on the acid-denatured state has been investigated by circular dichroism (CD), fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show considerable accumulation of secondary structure when the acid-denatured bromelain is subjected to 70% (v/v) TFE and exhibited close resemblance to spectral features of those of pH 7.0 preparation. Interestingly, the acid-denatured state also regained some tertiary structure/interactions, with increasing concentration of TFE and at 60% (v/v) TFE, these approached almost those of the native like state. However, further increase to 70% (v/v) TFE resulted in complete loss of tertiary structure/interactions. Tryptophan fluorescence emission studies also suggested the induction of significant compact structure at 60% (v/v) concentration of TFE. In addition the acid-denatured state showed enhanced binding of ANS in presence of 60% (v/v) TFE. Taken together these observations suggest the existence of a molten globule state in acid-denatured bromelain between 60 and 70% (v/v) TFE. A similar molten globule state under identical conditions has been identified in reduced and carboxymethylated preparation of stem bromelain as reported in our earlier communication [Arch. Biochem. Biophys. 413 (2003) 199]. Comparison suggests unfolding/folding behavior of the bromelain to be independent of the intactness of the disulfide bonds.
Gupta
Pawan
P
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India. pawan_g75@hotmail.com
Khan
Rizwan H
RH
Saleemuddin
M
M
D000143:Acids / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D001963:Bromelains / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D014270:Trifluoroethanol / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D000143:Acids; D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol; D014364:Tryptophan; D001963:Bromelains; C091760:stem bromelain; D019791:Guanidine