The Effect Of Hydroxylation On The Thermal Stability Of Collagen

Collagen is a bio-macromolecule that synthesized by animal cells. As a biomaterial, collagen exhibits biodegradability and has weaker antigenecity and better biocompatibility than other natural polymers. It is one of most common biomaterials with broad applications ranging from drug delivery to tissue engineering, scaffolds, cell culture and materials for wound dressing. However, the relatively fast biodegradation rate and low thermo and mechanical strength of these biomaterials obtained from collagens directly have been one of the crucial factors that limit the use of collagens.This study based on the thermal stability of collagen, investigating the relationship between hydroxyl modification and thermal stability with collagen. By analyzing the internal structure of collagen, the thermal stability and mechanical properties of collagen are mainly influenced by the interaction between three α chains and hydroxyl modification effect. Mainly studied are as following aspects;1. This study is focused on the collagen extracted from BN rats skins in different weeks, using acid formulation extract the collagen in BN rats skins, purified the collagen by gel filtration chromatography, the collagen molecular weight is about 300 kDa, usually its diameter is 1.5 nm, the linear length of molecular is about 300 nm. In the gel filtration chromatography, the retention time for the aim component is about 10 min. Using SDSPAGE to determine the components molecular weight that collected from the gel filtration chromatography, the collagen after refined shows three obvious main lane in SDS-PAGE, the molecular weight of 100 kDa stripe for collagen is α chains, the molecular weight of 200 kDa stripe for collagen is β chains, the molecular weight of complete collagen is 300 kDa stripe. Results indicate that the refined collagen triple-helix structure is complete, the protein structure is stable.2. Experiment investigated the influence of Hyp content on the thermal stability of collagen, chromatographic conditions: ZOBARX-C18 reverse-phase chromatographic column(2.1 x 150 nm, 5 microns), mobile phase A: 0.05 mol/L sodium acetic acid aqueous solution, mobile phase B: acetonitrile-water=1:1(V/V), detection wavelength is 360 nm. At first, extraction of collagen from 1, 2, 4, 5, 8 weeks of BN rats skin were determined, the method of amino acid composition analysis for Hyp content in different growing rats references were 33.5 ‰, 88.9 ‰, 99.3 ‰, 99.6 ‰, 99.7 ‰. Through the amino acid composition analysis for BN rat skins, with the growth of the BN rat, hydroxyproline in the skin tissue is gradually rise, then stabilizing.3. The influence of hydroxyproline content on the denaturation process of collagen were investigated using the differential scanning calorimeter(DSC) and the circular dichroism(CD) spectroscopy. The CD spectra indicated that collagen obtained from rat skin had the secondary structure typical for collagen. The helix content decreased when denatured by heat treatment. The denaturation temperature and molar enthalpy change were determined using DSC. The results show that the triple-helix become disordered at 41.3 ℃(Tm). The molar enthalpy change increased with quantity of hydroxylation of proline. CD spectrum manifested that part of the triple-helix changed into random coil structure when the denaturation temperature was higher than 41.3 ℃. The hydroxylation of proline modification is the key factor affecting on the structure of collagen during denaturation process.4. The application analysis the collagen viscosity, its elastic modulus G ’ increases with the increase of BN rats growth period, this is because the G’ means that the change of protein structure, when the structure change, collagen de-polymerization of triple helix structure.This experiment was studied the relationship in the collagen structure of Hyp content and enthalpy change by thermal induced changes. SDS-polyacrylamide gel electrophoresis analysis the refined collagen, consistent with the theory of collagen molecular weight. By DNFB reverse-phase chromatographic determination of amino acid composition in the extract collagens, the collagen content of Hyp and Gly in consistent are based on the calculation result of amino acid sequence, it shows that the collagen extracted in rats is type I collagen. Application of DSC was carried out on the extracted collagen Tm and molar enthalpy change detection, in which the total enthalpy change can be made of triple-helix structure of collagen de-polymerization of structure change. Firstly, from the molar enthalpy change of collagen, its own structure change of molar enthalpy change, and proline hydroxylation modification caused by hydrogen bond enthalpy change required. The samples are based on rat references on extraction, the polypeptide chain structure and amino acid composition are consistent, so the main reason for the differences in enthalpy change is caused by hydrogen bonding effect of Hyp, analyzing the relationship between the hydroxyproline content in BN rats skin of different weeks and critical denaturation temperature and the enthalpy change, the Hyp factors is the key to improve the stability of three helical structure of collagen, to further explore the proline hydroxylation modification sites on its stability provides basic research.