Title
Differential behaviorof native and denatured collagen in the presence of alcoholic solvents: A gateway to instant structural analysis
Author
Arun Gopinath, Ganesh Shanmugam, Balaraman Madhan, Jonnalagadda Raghava Rao
Year
2017
Journal
International Journal of Biological Macromolecules
Abstract
We report a novel method that exploits the differential solubility properties of native and denatured proteins to distinguish between them. We chose to study this using collagen because its unique native triple helical structure is critical for the desired properties of collagen-based biomaterials. We found that native and denatured collagen separate out from solution in alcohols(methanol, ethanol, n-propanol and isopropanol), but as different phases. Native collagen undergoes tight gel-like separation(macrophase) from the solution in alcohols(90%,v/v), whereas denatured collagen separates as particles(microphase), which turn milky-like turbid. It was found that the pH of the medium had to be alkaline for turbidity formation, which necessitated the use of buffered alcohols. In solutions having mixtures of native and denatured collagen, the method has sensitivity to visually confirm the presence of native collagen even at ratios as low as 10%. It was also confirmed that the formation of turbidity was a direct function of the concentration of denatured collagen. A thermal denaturation experiment, wherein stages of denaturation were studied both by the proposed method and circular dichroism, showed that information obtained from both methods of analysis was comparable. This highlights the potential of the proposed method to become an instant foolproof test for collagen.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Chemical stability, Protein folding, Protein denaturation, Thermal stability, Thermodynamics, Biochemistry