Studies On The Effect Of Heme On The Local Conformation Of Myoglobin

Myoglobin(Mb) is the protein which is studied thoroughly on the structure and function. Two components constitute Mb, globin and the prosthetic group (Heme).Globin has eightα-helix,small amount ofβ-turn and random coil. The function of Mb and the structural interaction between globin and Heme have been reported clearly.Amyloid fibril is a one-dimentional crystal-like proteinaceous aggregation, which is rich inβ-sheet. In 2003, Fandrich et al.found that apoMb is able to form amyloid fibril easily in pH9 solution including 50mM sodium borate at 55℃-75℃.We studied on the effect of Heme to the local conformation of myoglobin and got the spectrums of both Mb and apoMb by DSC, CD ,ThT fluorescence and Limited Proteolysis at 25,31,37,43,49℃.The results show:there is only one peak in the DSC spectrum of Mb(Tm=73℃). there are two peaks in the DSC spectrum of apoMb(Tm1=61℃,Tm2=83℃). The first peak of apoMb is a character of conformation changing. The second peak of apoMb is a character of unfolding of amyloid fibril.As the temperature rises,αhelix is destroyed, random coil increases.the intensity of 208nm and 222nm of the CD spectrum of Mb and apoMb decreases in a regular way. the intensity of 208nm and 222nm of the CD spectrum of Mb decreases in a linear equation way.however, the intensity of 208nm and 222nm of the CD spectrum of Mb decreases in a two-linears equation way. There is a inflexion arising near 37℃αhelix is destroyed quickly after this inflexion temperature . As the temperature goes up, the ThT fluorescence intensity of Mb don't change at 25,31,37,43,49℃which shows Mb don't form amyloid fibril at the five temperatures.However, the ThT fluorescence intensity of apoMb increase gradually.that means apoMb form amyloid fibril when the temperature rises. The ThT fluorescence intensity of apoMb at the the five temperatures is much lower than its intensity at 65℃. This result indicate:apoMb is hard to form amyloid fibril when the temperature is lower than 49℃.because heme still has a strong ability to stabilize the local conformation of myoglobin at this temperature below 49℃.At 25,31,37,43,49℃, both the limited proteolysis kinetic curves of Mb and apoMb are linear equation. The velocity of apomb is higher than Mb. However the difference of velocity between Mb and apoMb decreases gradually along with the increase of temperature. There is a inflexion arising near 37℃. The result shows that heme makes contribution to the thermal stability of holoMb but effect is temperature-dependent.the ability of stabilizing holoMb decreases gradually when the temperature goes up over 37℃.From the whole results above and the primary structure information of amino acids of Mb,we get two deductions . 1)Heme stabilizes the monomer structure of Mb and inhibites the assemble function of monomers by inhibiting the local vibration of A helix. 2)There is a interaction area in one side of A helix,this area is related to the formation of amyloid fibril of Mb.