Surfactant-mediated amyloidogenesis behavior of stem bromelain; a biophysical insight.

Neurodegenerative disorders are mainly associated with amyloid fibril formation of different proteins. Stem bromelain (SB), a cysteine protease, is known to exist as a molten globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the gut epithelium of intestine upon oral administration. Protein-surfactant complexes are widely employed as drug carriers, so the nature of surfactant toward protein is of great interest. The present work describes the effect of cationic surfactants (CTAB & DTAB) and their hydrophobic behavior toward amyloidogenesis behavior of SB at pH 10.0. Multiple approaches including light scattering, far UV-CD, turbidity measurements, and dye binding assay (ThT, Congo red and ANS) were performed to measure the aggregation propensity of SB. Further, we monitored the hydrodynamic radii of aggregates formed using dynamic light scattering technique. Structure of fibrils was also visualized through fluorescence microscopy as well as TEM. At pH 10.0, low concentration of CTAB (0-200 µM) induced amyloid formation in SB as evident from a prominent increase in turbidity and light scattering, gain in ß-sheet content, and enhanced ThT fluorescence intensity. However, further increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast, DTAB did not induce fibril formation at any concentration used (0-500 µM) due to lower hydrophobicity. Net negative charge developed on protein at high pH (10.0) might have facilitated amyloid formation at low concentration of cationic surfactant (CTAB) due to electrostatic and hydrophobic interactions.

Concentration-dependent antagonistic persuasion of SDS and naphthalene derivatives on the fibrillation of stem bromelain.

Sodium dodecyl sulfate, a biological membrane mimetic, can be used to study the conversion of globular proteins into amyloid fibrils in vitro. Using multiple approaches, the effect of SDS was examined on stem bromelain (SB), a widely recognized therapeutic protein. SB is known to exist as a partially folded intermediate at pH 2.0, situation also encountered in the gastrointestinal tract (its site of absorption). In the presence of sub-micellar SDS concentration (500-1000 µM), this intermediate was found to exhibit great propensity to form large-sized ß-sheeted aggregates with fibrillar morphology, the hall marks of amyloid structure. We also observed inhibition of fibrillation by two naphthalene-based compounds, ANS and bis-ANS. While bis-ANS significantly inhibited fibril formation at 50 µM, ANS did so at relatively higher concentration (400 µM). Alcohols, but not salts, were found to weaken the inhibitory action of these compounds suggesting the possible involvement of hydrophobic interactions in their binding to protein. Besides, isothermal titration calorimetry and molecular docking studies suggested that inhibition of fibrillation by these naphthalene derivatives is mediated not just through hydrophobic forces, but also by disruption of π-π interactions between the aromatic residues together with the inter-polypeptide chain repulsion among negatively charged ANS/bis-ANS bound SB.