Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form.
We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.
Ahmad
Basir
B
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India.
Khan
Rizwan Hasan
RH
D031230:Ananas / Q000201:enzymology*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001963:Bromelains / Q000737:chemistry*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D010940:Plant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D001963:Bromelains; C091760:stem bromelain