The Research To The Effect To Myoglobin And Its Mutants By Different Classes Of Surfactants

The interaction between surfactant and protein has an important theoretical meaning and a great practical value. In this paper, the mechanism and the effect on the structure and function of myoglobin(Mb) and its mutants (Mb(D60K) and Mb(K56D)) by different classes of surfactants were investigated by means of spectroscopies and stopped-flow fluorescence technology under simulative physiological conditions. Investigations as follows:1. Surfactant is an amphiphilic material:one side is hydrophilic group and the opposite end is hydrophobic group. According to the dissociation properties of the hydrophilic group, surfactant can be divided into anionic, cationic, zwitterionic and nonionic surfactant. Myoglobin is a kind of polyelectrolyte with senior structure. Its electric properties changes as a result of changes in external conditions. So the mechanism of interaction between surfactant and myoglobin is more complicated. The complexes of Mb with anionic surfactants sodium bis (2-ethylhexyl) sulfosuccinate (AOT) and sodium dodecyl benzene sulfonate (SDBS), with cationic surfactants dodecyl trimethylammonium bromide (CTAB) and dodecyltrimethyl ammonium bromide (DTAB), and with zwitterionic surfactant propanesulfonate (CHAPS) were investigated by the method of spectroscopy techniques. The analysis turns up that anionic and cationic surfactants can interact with Mb by means of electrostatic and hydrophobic forcing. They alter the microenvironment of porphyrin in the centre of the Mb. Under simulated physiological conditions, Mb is negatively charged, so cationic surfactants have a stronger effect with affecting the heme center directly, can induce heme monomer to leave the hydrophobic cavity of Mb by electrostatic attraction mainly. Interaction between surfactant and protein depends on surfactant concentration. No interaction was detected between zwitterionic surfactant and Mb in a large range concentration because of the neutral charge of CHAPS.2. A single amino acid change of a protein can cause its structural and functional changes. The mechanism and difference from Mb of reaction process between Mb(K56D) and three classes of surfactants were investigated. The results show that Mb(K56D) solution contains MbFe2+-O2and MbFe3+-H2O protein forms. Surfactants interact with both of them. The ways to chang the porphyrin coordination are all the same to Mb. Fluorescence and CD spectroscopy show that site-direct mutagenesis has an effect on the protein structure in large extent, but has little effect on the overall charge of the protein under simulative physiological conditions.3. The mutation sites of Mb(D60K) and Mb(K56D) are close, but the amino acids have the opposite charge. Different mechanisms of intereaction between three kinds of surfactants and D60K were studied. According to the experimental results, the reaction process of Mb(D60K) and surfactant is the same as the Mb complex. Amino acid60changed from Asp to Lys also has little effect on the overall charge of the protein, but can change the structure of Mb, a-helix content decreases as shown in the fluorescence and CD spectroscopy results.4. Dynamic curve can give a reasonable explaination to a reaction mechanism. By stopped-flow fluorescence spectroscopy, the reaction kinetics between SDBS/DTAB and Mb/Mb(D60K) was investigated. It was found that the reaction was a second-step reaction. The rate constants of Mb/Mb(D60K) and SDBS/DTAB both decreased with the surfactants concentration increasing at30℃, which indicated that concentration of Mb or Mb(D60K) deceased in the reaction by dimerized in order to become more stable. Mb(D60K) surface has one more lysine than Mb, and it tends to internal polymerization, which leads to less dimerization between protein molecules, so the k value of its reaction with SDBS or DTAB reduced in a smaller degree compared to Mb.