ABSTRACT
Plants under stress need to favour certain pathways so as to survive the stress period. Protection of specific enzymes by proline and other osmolytes could be one such mechanism to favour some pathways/processes. Therefore, the influence of osmolyte proline on conformational changes of various proteins caused by hydrogen peroxide (H2O2) was studied by intrinsic and extrinsic fluorescence emissions. H2O2 caused conformational change in proteins. Results indicated that for Alcohol dehydrogenase (AD) and Glutamate dehydrogenase (GD) enzymes, H2O2 induced conformational change was high and that for Glucose 6-phosphate dehydrogenase (G6PDH) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was low. Fluorescence and far-UV, CD measurements of catalase demonstrated that the H2O2 stabilized the protein secondary structure at low concentrations but destabilized it at higher concentrations. Intrinsic and ANS fluorescence results showed that proline at a concentration of 1.0 M prompted a reduction in the H2O2-induced exposed hydrophobic surfaces of studied enzymes, to different degrees which suggests its differential protective effect. Furthermore, SDS-PAGE studies revealed that proline was not able to reduce or inhibit the H2O2 mediated aggregation of GAPDH.
ABSTRACT
The binding mechanism between pterostilbene ( PTE) and human serum albumin ( HSA) was investigated by fluorescence spectrometry and surface enhanced Raman spectroscopy (SERS) under simulated physiological conditions. The experiment result showed that the effect between PTE and HSA was a static fluorescence quenching with F?rsterˊ s non-radioactive energy transformation, and PTE could bind HSA strongly with a 1: 1 molar ratio. The binding distances between PTE and HSA was 1. 495 nm, and the binding constants (KA) between PTE and HSA were 1. 12 × 104 (298 K), 4. 07 × 104 (304 K) and 2. 45 × 105 L/ mol (310 K). SERS revealed that PTE combined with HAS by methoxy group. Thermodynamic data indicated that the interaction between PTE and HSA was mainly hydrophobic interaction. Marker competition experiments pointed out that the primary binding site for PTE was located at site Ⅲ in HSA. Three-dimensional, synchronous fluorescence spectrum and SERS showed that the conformation of HSA changed apparently with the addition of PTE, resulting in the tryptophan residue of HSA exposing to a less hydrophobic micro-environment. However, the conformation of PTE did not change apparently with the addition of HSA.