Characterization of a partially folded intermediate of papain induced by fluorinated alcohols at low pH.
A systematic investigation of the effects of aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) on the structure of acid-unfolded papain (EC. 3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits substantial secondary structure as beta-sheet and is relatively less denatured as compared to 6 M guanidine hydrochloride (GdnHCl) but loses the persistent tertiary structure of the native state. Addition of HFIP and TFE caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Induction was 20% more in HFIP than TFE. Interestingly, at 13% (v/v) HFIP and 30% (v/v) TFE a near-UV CD spectrum approaches the native-like spectral features. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on the addition of HFIP and TFE to acid-unfolded papain. Maximum ANS binding occurs at 13% (v/v) HFIP and 30% (v/v) TFE, suggesting a compact "molten globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in presence of 13% (v/v) HFIP and 30% (v/v) TFE showed the recovery of enzymatic activity by 54 and 61%, respectively. Thermal stability of these states was assessed by changes in fluorescence emission maximum and absorbance at 292 nm. Temperature-induced unfolding of papain at pH 2 was non-cooperative and the transition curves were biphasic in nature. Temperature-induced unfolding of HFIP and TFE-induced state was weakly cooperative in comparison to cooperative transition of native.
Naeem
Aabgeena
A
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Khan
Khursid Alam
KA
Khan
Rizwan Hasan
RH
D000438:Alcohols / Q000737:chemistry* / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D029441:Carica / Q000378:metabolism; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010206:Papain / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D014270:Trifluoroethanol / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D000438:Alcohols; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol; D014364:Tryptophan; D010206:Papain