Wide-angle X-ray scattering as a probe for insulin denaturation.

Wide-angle X-ray scattering (WAXS) from lyophilized protein is characterized by the presence of two relatively broad scattering peaks that are linked to protein structure. This work is concerned with the possibility of utilizing these peaks in the probing of the unfolding and breakdown of insulin. Native insulin is subject to thermal denaturation in the presence and in the absence of thiol catalysts. Denatured products are acid-trapped, lyophilized and monitored using WAXS in addition to Fourier transform infrared spectroscopy (FTIR), gel filtration chromatography and Transmission Electron Microscopy (TEM) as supportive techniques. Results show that the WAXS peak at a d-spacing about 10 A is sensitive towards the alpha-helix content of insulin. A reduction in the intensity of such peak is proven to be directly linked to the reduction of native insulin having normal alpha-helix content. The supportive techniques confirmed the decrease in the alpha-helix content of insulin which accompanied the different denaturation treatments. Copyright 2010 Elsevier B.V. All rights reserved.