Model Of Structural Elements Applied To Analyze The Stability Of Enzyme

Studies on the mechanism of protein folding have been attracting the attention of researchers in the fields of biochemistry and molecular biology at present,which play an important role in gaining an understanding of the relationship between the structure of proteins and their functions.There are many methods to determine the process of unfolding of protein,but it is difficult to directly capture the characteristics of structure and species distribution of the transition state.The unfolding curves of enzymes with catalytic function are significantly different of activity characterization and spectroscopic characterization.Molecular dynamics(MD)restricted by simulation time and computer performance only can be used to study protein which is molecular weight less than 100 kD.In view of this,it is urgent to develop a new approach to overcome the defects.This paper adopted the uv-vis spectroscopy,fluorescence spectroscopy,CD and fluorescent lifetime to study the unfolding process of lysozyme and bovine carbonic anhydrase.The unfolding mechanism of enzyme are given combining with structural primitive model.The results of guanidine hydrochloride and reduction of urea induced unfolding of lysozyme molecules show that:The results of unfolding of lysozyme molecules induced by guanidine hydrochloride and reduction of urea show that:the unfolding process of lysozyme conformed to "three state model" and "continuous reaction" mechanism;in the concentration of 0?3.8 mol/L guanidine hydrochloride,?-piece structure domain(enzyme active site)of lysozyme unfolded firstly,while?-helix unfolded in 3.8?5 mol/L;the application of structure element model not only described the whole unfolding process of lysozyme molecules accurately,but also reported more reasonable species distribution within the scope of all denaturant concen-tration.The experimental results of guanidine hydrochloride induced bovine carbonic anhydrase unfolding under the different temperature show that:high temperature was unfavorable to chemical reaction,hydrophobic interaction played a dominant role in reaction;At 25?,the unfolding process of bovine carbonic anhydrase conformed to "three state model" and "continuous reaction" mechanism.With the increase of concentrations of guanidine hydrochloride,fluorescence spectrum showed that the environment of tryptophan of BCA changed from nonpolar to polar.CD spectra showed that secondary structure of?-helix of BCA undergone the course of reducing firstly then unchanging and finally disappeared,a-helix did not change when the BCA residual activity experienced from two to single transition.The experimental results of temperature induced lysozyme,BCA and apo-BCA showed that:thermal denaturation of the lysozyme accorded with the "four state ",while renaturation belonged to the model of "two state".The process of thermal denaturation and renaturation were confirmed without the formation of aggregation applying fluorescence resonance light scattering(RLS),ThT fluorescence spectrum method,the Native-SDS;while BCA tended to aggregate at the same conditions;apo-BCA was more likely to aggregate than BCA and the rate of aggregation was about seven times of the BCA.We made use of the "parallel reaction","continuous reaction" two kinds of reaction mechanism to study the species distribution in the process of unfolding of enzyme firstly.Using structure primitive model to solve the problem of inconsistency between enzyme activity and spectrometry.