The Research To The Interaction Between Hemoproteins And NO By Spectroscopic Technology

The coordination reaction between hemoprotiens with small ligands exists extensively in life, People can better understand the structure and function of hemoprotein through the study on interaction small ligands with hemoprotein, and further excavating the application in medical treatments. This paper researched the reaction mechanism of cytochrome c and Myoglobin with NO using of ultraviolet visible(UV- Vis) absorption spectroscopy, fluorescence spectroscopy, circular dichroism(CD) spectra. The research contents in this thesis as flollows:⑴ Using UV-Vis spectra and CD spectra to study the interaction of wild type myoglobin Mb(WT), myoglobin mutant(Mb(D44K) and Mb(D60K)) and NO gas. The results showed that Mb(WT), Mb(D44K) and Mb(D60K) can be combined with NO, the ability to combine NO is Mb(D44K) > Mb(D60K) > Mb(WT), and protein structure will not be destroyed and still maintain its biological activity, with increasing of NO in a certain amount range. The coordination reaction occurs on NO and Fe of heme in the center of Mb(WT), the amino acid changes on the surface just affects its combination rate with NO.⑵ UV-Vis absorbance spectra was used to characterize hemoglobin(Hb), and then Hb spectras were analysised under different ionic strength and p H of buffers. It was favorable for the coordination reaction, when the 0.01 mol/L phosphate buffer solution was near neutral condition, the coordination reaction could reach equilibrium at the fastest speed.⑶ Using UV-Vis spectra, UV-Vis time course spectra, synchronous fluorescence spectra, CD spectra, to study the coordination reaction mechanism of different states Cyt c with proli NO/NO and its secondary structure changes. The results showed that Cyt c can react with NO produced by proli NO/NO directly under the condition of noting added. proli NO/NO adding into the Cyt c samples generated NO, entering the solution, the methionine(Met) on heme of Cyt c is to leave, then Cyt c combined with NO, at the same time, generating a new complexes cytochrome c-NO(Cyt c- NO). Cyt c- NO is unstable and will dissociate, the dissociation rate was 0.00711±0.00396 s-1. When the concentration of the proli NO/NO below 8.6×10-4 mol·L-1, 222 nm and 208 nm absorption peaks change little, and α-helix increases from 33.1% to 44.1%. Along with the increase of the concentration of NO, the secondary structure of Cyt c changes obviously, too much will destroy its secondary structure.In this paper, the reaction mechanism of the interaction of the hemoproteins(wild type two mutants, myoglobin and hemoglobin and horse heart cytochrome C) and the NO to understand and NO other types of heme protein, and other gases(CO, H2 S, etc.) provides the experimental basis, the mechanism of interaction between, And for the understand of the function of the NO and the hemoproteins, and the diseases’ diagnosis, treatment caused by the NO and the hemoproteins have the vital clinic significance.