Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH
✍ Scribed by Aabgeena Naeem; Sadaf Fatima; Rizwan Hasan Khan
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 2006
- Tongue
- English
- Weight
- 383 KB
- Volume
- 83
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
Abstract
A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS), hexa decyltrimethyl ammonium bromide (CTAB) and Tween‐20] 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 a substantial amount of secondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydrochloride) but loses the persistent tertiary contacts of the native state. Addition of detergents caused an induction of α‐helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Near‐UV CD spectra also showed the regain of native‐like spectral features in the presence of 8 m__M__ SDS and 3.5 m__M__ CTAB. Induction of structure in acid‐unfolded papain was greater in the presence SDS followed by CTAB and Tween‐20. Intrinsic tryptophan fluorescence studies indicate the change in the environment of tryptophan residues upon addition of detergents to acid‐unfolded papain. Addition of 8 m__M__ SDS resulted in the loss of ANS binding sites exhibited by a decrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. Maximum ANS binding was obtained in the presence of 0.1 m__M__ Tween‐20 followed by CTAB, indicating a compact “molten‐globule”–like conformation with enhanced exposure of hydrophobic surface area. Acid‐unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. These results suggest that papain at low pH and in the presence of SDS exists in a partially folded state characterized by native‐like secondary structure and tertiary folds. While in the presence of Tween, acid‐unfolded papain exists as a compact intermediate with molten‐globule–like characteristics, viz. enhanced hydrophobic surface area and retention of secondary structure. While in the presence of CTAB it exists as a compact intermediate with regain of native‐like secondary and partial tertiary structure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a molten globule state with tertiary folds. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 1–10, 2006
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