Investigation Of Temperature Stability Of Collagen And Bovine Serum Albumin By Terahertz Dielectric Spectroscopy

The biological function of proteins has close relation with the spaceconformation of amino acid residue, but the conformation prone to happeningchanges because of influence by ambient temperature. The research of the structurechanges and the deactivation mechanism of proteins are important to use of theprotein drugs safely during preparation, production, storage and transportation.Currently the detection technology is used to study of the structure and function ofproteins concludes chromatography and all kinds of spectrum method, etc. Althoughchromatography methods are simple and clear, they need complex pretreatmentprocess; Spectrum methods such as ultraviolet absorption spectrum, FTIR andRaman spectrum and so on, they usually can only reflect the inside information ofsingle molecule, and lack of ability providing the molecular conformation andinteraction information to define the function of proteins. The study found thatmolecular collective structure rotational energy of protein structure and conformationis located in THz wave band, we can get the complex dielectric spectrum of sampleby terahertz time-domain spectroscopy measurement, the dielectric spectrum candirect the molecular dipole polarization response which is close relation with themolecular structure. Terahertz dielectric spectrum is very sensitive to the changes ofstructure; we can understand the characteristic time of molecular movement andobtain a lot of information that is related to the structure and movement of molecularfrom the dielectric relaxation process. This paper studies the terahertz dielectricspectroscopy of solid collagen and bovine serum albumin in low temperature process,the heating process and heating degeneration; we use dielectric relaxation theory ofbiological molecules obtained the dielectric spectrum model, then get the dielectricrelaxation time and activation energy of proteins, and analyze the structure changesthat caused by the temperature.The woks in this paper mainly include:(1) The THz dielectric spectrum of the solid collagen and bovine serum albuminin low temperature and heating process was measured by THz-TDS. The Low temperature process used of liquid nitrogen modulating, and above the roomtemperature mainly depends on the electrical resistance heating. The temperaturecontrol range is90K~390K. We also measured these two proteins in natural state andafter heating state by THz-TDS in room temperature, the heating treatmenttemperature is110℃. Using of the differential scanning calorimetric (DSC)apparatus and FTIR measured the proteins of before and after thermal denatured.(2) Using dielectric spectrum theory established first-order Debye model, andfitting the terahertz dielectric spectrum of the solid protein, which in low temperatureand heating process. We get the corresponding dielectric relaxation time and find therelaxation time gradually increase as the temperature rising, this phenomenon iscorresponding with protein molecular internal conformational unfolding process thatis caused by the temperature changes. The changes of relaxation time meet with thetemperature dependency (Arrhenius equation) of the dielectric property, and obtainthe collagen’s activation energy are3.32×10~3J/(K· mol) and5.53×10~3J/(K· mol) inlow temperature and heating process; the bovine serum albumin’s activation energyare1.16×10~3J/(K· mol) and6.52×10~3J/(K· mol). This energy can not only as ameasure of the protein stability index, but also quantitatively represent the degree ofdifficulty of solid proteins in heating denatured process.(3) Using first-order Debye model fitted THz dielectric spectrum of the solidproteins in the natural state and after heating treatment state, and get their dielectricrelaxation time, we find the absorption and refractive index of the thermal denaturedproteins are reduced, and relaxation time is increased. The DSC curve of the heatingprotein has no endothermic peak, so we know the proteins take place completelydegeneration, while FTIR has not detected the structure changes of denaturedproteins. So they further prove that the THz dielectric spectrum is sensitive to thestructure changes of protein which is influent by temperature.