Specific molten globule conformation of stem bromelain at alkaline pH.
Stem bromelain (SBM), a therapeutic protein, is rapidly absorbed across the gut epithelium. Because SBM encounters an alkaline pH at its principal site of absorption, we investigated the alkaline-induced denaturation of SBM. From pH 7 to 10, the protein's secondary structure remained the same, although a slight loss of tertiary structure was observed. Above pH 10, there was a significant and irreversible loss of secondary and tertiary structure. At pH 10, SBM showed enhanced tryptophan fluorescence, however, the number of accessible tryptophans remained the same. The thermodynamics of temperature transition at pH 7 and 10 were strikingly different, with the former showing a two-phase transition endotherm, and the latter a broad non-two-state transition. At pH 10, SBM showed a significant increase in 8-anilino-1-naphthalene-sulfonate binding relative to the native state, suggestive of a specific molten globule (SMG) state. These studies suggest a distinct conformational rearrangement in SBM, at the protein's isoelectric point.
Dave
Sandeep
S
Institute of Microbial Technology (CSIR), Chandigarh 160036, India.
Mahajan
Sahil
S
Chandra
Vemika
V
Dkhar
H Kitdorlang
HK
Sambhavi
Gupta
Pawan
P
D031230:Ananas / Q000201:enzymology; D001963:Bromelains / Q000737:chemistry* / Q000493:pharmacokinetics; D002942:Circular Dichroism; D019791:Guanidine; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007408:Intestinal Absorption; D007526:Isoelectric Point; D008517:Phytotherapy; D010940:Plant Proteins / Q000737:chemistry* / Q000493:pharmacokinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D010940:Plant Proteins; D014364:Tryptophan; D001963:Bromelains; C091760:stem bromelain; D019791:Guanidine