Studies On The Unfolding And Refolding Of Arginine Kinase

Arginine kinase (ATP: L-arginine phosphotransferase EC 2.7.3.3), plays an important role in cellular energy metabolism in invertebrates. Arginine kinase from shrimp (Feneropenaeus Chinensis) was taken as a model protein to study the unfolding process and effects of the induced helical structures on refolding.The denaturation of arginine kinase (AK) in various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) has been investigated by following the methods of far-ultraviolet circular dichroism (CD) spectra, intrinsic fluorescence, ANS binding and activity assay. The concentration of HFIP employed was under 10% (v/v) to avoid the aggregation of protein. In 0.5%-1.5% HFIP solutions, no remarkable changes of secondary structure and fluorescence emission maximum of AK have been observed, while the ANS increased obviously. Further addition of HFIP to 6% caused a significant red shift of the emission maximum and the activity of enzyme totally lost. Comparison of inactivity and unfolding process showed that marked inactivation occurred before significant conformational change of AK. The changes of intrinsic fluorescence and ANS intensity showed that there existed a relatively stable molten globule state of AK in the HFIP solution ranging between 0.5% and 1.5% (v/v) and the molten globule state may be a native-like unfolding equilibrium intermediate .The addition of hexafluoroisopropanol (HFIP) considerably induced α-helices in urea-denatured arginine kinase (AK). Folding kinetics studies showed that the folding rates of the urea-denatured AK were significantly decelerated after being induced in various concentrations of HFIP. The deceleration of folding did not correlate linearly with the HFIP concentration and a minimal folding rate appeared as the inducing HFIP concentration was 5% (v/v). When the inducing concentration of HFIP increased to 10% (v/v), the folding rate also increased to about 60% of...