The Mechanism Of Heavy Metal Hg～+ Acting On Serum Albumin

There are numerous proteins in organisms which are macromolecules containing multiple ligands. The side chains and terminal residues of many proteins have a potential ability to bind most metal ions. Serum albumin, whose physiological function is well correlated with its particular structure, is involved in the transportation and storage of both exogenous and endogenous substances, and also plays a significant role in carrying, buffering and nutrition. So far the studies of protein probe mostly focus on organic micro-molecules such as analytical reagents, however, there are relatively much fewer researches by metal-ion protein complex probe. When entering into animal bodies, heavy metal Hg+ acts on proteins, which may effect or change their intrinsic structure or function. In order to probe into the interaction between Hg+ and proteins, as well as the in vivo mechanism of Hg+ metabolic pathway and biological effect, in this work, the effect of spectral character change by Hg+ interaction on BSA and HAS, the specific modification on trptophan residue of those two proteins by NBS, the difference between such modifications acting on Hg+ binding, and the comparison of Hg+ influence before and after NBS treatment are studied by means of UV differential spectra, emission fluorescence and circular dichroic measurements and SDS PAGE as well.At first we examined the effect of BSA/HSA absorbing spectra by HgCl2 treatment without buffer system. The result shows that Hg+ can lead to the change of BSA UV absorbing spectra, and both the intensity and location of absorbing peakchange as the treating time increases. Then BSA and HSA were chemically modified by NBS. dialyzed over night and UV differential spectroanalysed. The result suggests that, compared with serum albumin solution without Hg+, the UV spectra of Hg+-serum albumin system (3:1 molar ratio) shows a distinct LMCT (ligand-to-metal charge transition) band and the LMCT of Hg+-BSA system has a slight decrease at 235nm as time increases. After 6h it no longer decreases, however, the LMCT of Hg+-HSA system increases as time goes by. Further research indicates that LMCT can also be detected when Hg+-BSA/HAS systems (3:1 molar ratio) were modified by NBS, yet compared with non-modified BSA, the LMCT of modified Hg+-BSA shows a more distinct change as time increases. Excited respectively at 278nm and 295nm, two kinds of serum albumin emission fluorescence spectra show that the fluorescent peak of NBS modified BSA/HSA has an obvious blue shift when excited at 278nm and the intensity is 1/10 of the non-modified; the NBS modified BSA remains less than 2 % fluorescence when excited at 295 nm and has a blue shift as well, which indicates that Trp residue has been modified. Hg+ also cause the intrinsic fluorescence change of the two serum albumins and the intensity of BSA emission peak gradually falls as time increases, but the one of HSA decreases at first and enhances later. Non-denaturing polyacrylamide gel electrophoresis suggests that the transferring rate of NBS modified BSA decreases compared with non-modified, but Hg+ treatment has relatively little influence on non-modified and NBS modified HSA, except that the protein band broadens a little. The CD spectra measurement shows that the near-UV region of non-modified HSA and BSA both have typical a -helix and Hg+ treatment has apparent effect on configuration so that the secondary structure decreases distinctly. However. Hg+ has little effect on near-UV CD spectra of BSA. which indicates little change of secondary structure, but at the same time, NBS modification has some influence on far-UV structural configuration and Hg+ also changes the configuration markedly. The fact that Hg+ treatment has different effect upon spectra of BSA and HSA is probably relative to the structural difference of the two proteins.