Study On Physicochemical Properties And Structure Of Collagen From Golden Pompano(Trachiturus Ovatus)Skins

Golden pompano was one of largest marine fishes farming in China,80%of them are used for producting fish balls and fish fillets.During the process,it always have a lot of wastes like fish skins.Therefore,acid-solubilized collagen?ASC?and pepsin-solubilized collagen?PSC?from golden pompano?Trachiturus ovatus?skins were extracted and physicochemical properties of collagen were investigated in this study.Then,?₁-subunit,?₂-subunit and?-subunit were isolated from acid-solubilized collagen by carboxymethyl cellulose?CM-52?column chromatography and the relationship between the primary structure and the physicochemical properties of collagen and subunits were investigated.In addition,effects of pH on the properties of collagen fibril were investigated.Firstly,acid-solubilized collagen?ASC?and pepsin-solubilized collagen?PSC?from golden pompano skins were extracted and characterized.The molecular weight of ASC was about 130kDa for?₁ and 115 kDa for?₂,which were slightly higher than those of PSC.Similar amino acid composition and fourier transform infrared?FTIR?spectra were observed in both collagens,but slight differences were found in the peptide maps of collagen digested by V8 protease and trypsin.The denaturation temperatures?Tds?of ASC and PSC calculated from the reduced viscosity were31.8oC and 30.0oC,while the transition temperature?Tm?of ASC and PSC analyzed by DSC were33.0oC and 32.0oC.ASC has a lag phase,a growth phase and a plateau phase in the turbidity-time curve,while PSC has not similar phenomenon.It was found that the fibril gel of ASC could be formed at 25oC,leading to improve the thermal stability.The effect of pH on the properties of collagen fibril from golden pompano skins was studied.The results showed that the isoelectric point of collagen was 5.27.Results of turbidity and rheological viscoelastic revealed that the assembly rate of collagen increased first and then decreased with the increase of pH,and the rate was the highest at pH 5 and lowest at pH 7.According to the results of scanning electron microscope?SEM?,fibril diameter of pH5,6,7 and8 were 30-34 nm,41-45 nm,50-57 nm,60-71 nm,respectively,and clear D-period was found in SEM pictures of pH 7 and pH 8.The gel strength and DSC of collagen fibril gel showed that the bigger the pH,the greater the strength and thermal stability.The above results showed that fibril-forming ability of collagen increased with the increase of pH.At last,alpha and beta subunits were isolated from acid-solubilized collagen of golden pompano skins by carboxymethyl cellulose?CM-52?column chromatography,and the cDNA sequences of?₁ and?₂ were cloned from golden pompano skins and the deduced amino acid sequences were analyzed.The?₁ and?subunits were successively eluted from CM-52 column by a linear gradient of 0-0.035 M NaCl in the same pH of 4.7?0.050 M sodium acetate buffer?,while?₂-subunit was eluted by roughly the same NaCl concentration in the pH of 5.2.SDS-PAGE result showed that the molecular weights of?₁,?₂ and?subunits were about 131,119 and 256 kDa,respectively.Two endothermic peaks were found in the DSC curve of?subunit,and the thermal transition temperature?Tm?of?₁ was higher than that of?₂.Based on the results of DSC and CD, it was found that the thermal stability of collagen subunits was associated with their secondary structures.Analyses of deduced amino acid sequences revealed that theoretical sizes and isoelectric points of collagen?₁ and?₂ were 137 kDa,pH5.95 and 126 kDa,pH9.27,respectively.Compared to the collagen?₂,collagen?₁ had a cysteine-rich globular domain and more tripeptide unit of Gly-Pro-Pro.Furthermore,?-helix in the collagen?₁ was found to have specificity in the different species.It is concluded that?₁ subunit plays more important role in the physicochemical properties of collagen than?₂ subunit.In conclusion,the thermal stability and the fibril-forming ability of ASC was higher than those of PSC,which could be attributed to the complete primary structure including telopeptide region.Fibril-forming ability of collagen was strongest in pH 8.Alpha and beta subunits were isolated from acid-solubilized collagen of golden pompano skins by carboxymethyl cellulose?CM-52?column chromatography by controlling the ionic strength and pH.There are differences between the three subunits in the thermal stability and the secondary structure.Comparing the primary structure of?₁ and?₂,?₁ played an important role in the thermal stability and fibril properties of collagen.