Influence of salts and alcohols on the conformation of partially folded intermediate of stem bromelain at low pH.
The effect of salts and alcohols was examined on the partially folded intermediate (PFI) state of stem bromelain reported at low pH (Haq, Rasheedi, and Khan (2002) European Journal of Biochemistry 269, 47-52) by a combination of optical methods like circular dichroism, intrinsic fluorescence and ANS binding. ESI mass spectrometry was also performed to see the effect, if any, on the overall tertiary structure of the protein. Increase in ionic strength by the addition of salts resulted in folded structures somewhat different from the native enzyme. Salt-induced intermediates are characterized by increase in helical content and a significantly reduced exposure of hydrophobic clusters relative to the state at pH 2.0. The emission wavelength maximum of intrinsic fluorescence was shifted towards that of native enzyme. ESI-MS data show decreased accessibility of ionizable/protonation sites suggestive of a folded structure. On the other hand, alcohol-induced intermediates though exhibiting increased helical content are apparently largely unfolded as observed by ESI. Thermal denaturation of a representative intermediate, each from the group of salts and alcohols examined, was also performed to check their relative stabilities. While the alcohol-induced state showed a cooperative thermal transition, the salt-induced state shows non-cooperative thermal denaturation.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Soghra Khatun
SK
Haq
Sheeba
S
Rasheedi
Pallavi
P
Sharma
Basir
B
Ahmad
Rizwan Hasan
RH
Khan
D000438:Alcohols / Q000737:chemistry; D031230:Ananas / Q000201:enzymology*; D001963:Bromelains / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012492:Salts / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D000438:Alcohols; D010940:Plant Proteins; D012492:Salts; D001963:Bromelains; C091760:stem bromelain