ISOLATION, PURIFICATION, AND CHARACTERIZATION OF BOVINE TENDON COLLAGEN AND ANALYSIS OF GLYCINE, PROLINE, AND HYDROXYPROLINE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-FLUORESCENCE
DOI:
https://doi.org/10.22159/ijap.2018.v10s1.69Keywords:
Amino acid, Bovine tendon collagen, Content, Derivatization, Fluorescence, Glycine, High-performance liquid chromatographic, Hydroxyproline, Optimization, ProlineAbstract
Objective: In this study, collagen isolated from bovine tendon was purified and characterized, and the optimum conditions for analysis of glycine,
proline, and hydroxyproline were determined.
Methods: The collagen isolation process used 0.1 N NaOH as a pretreatment, 0.5 M acetic acid in the extraction, 0.9 M NaCl in the salting-out
step, centrifugation and dialysis for purification, and freeze-drying as the final step. The characterization of the collagen included analysis of the
organoleptic properties, pH, moisture content, viscosity, and ash content. A Fourier-transform infrared (FTIR) spectroscopy analysis and Casson's
trichrome staining were also performed. The collagen was hydrolyzed in 6 N HCl for 24 h and derivatized using 9-fluorenylmethoxycarbonyl chloride.
The optimum condition was conducted from the optimal wavelength, selection of mobile phase composition, and flow rate.
Results: The average content was 11.867±0.20% for glycine, 33.247±0.20% for proline, and 10.51±0.23% for hydroxyproline. The optimum condition
analysis for collagen was achieved by high-performance liquid chromatography (HPLC) with a C18® column and a fluorescence detector (excitation:
265 nm and emission: 320 nm) with mobile phase acetate buffer (pH 4.2):acetonitrile (55:45), and the flow rate was 0.8 mL/min.
Conclusion: The collagen isolated from bovine tendon was obtained at a yield of 0.690%, and the identity was confirmed by FTIR functional group
analysis and Casson's trichrome staining. The HPLC conditions using a fluorescence detector for analysis of glycine, proline, and hydroxyproline
concentrations in the bovine tendon collagen were optimized. The analysis of amino acids gave the average levels of 33.247±0.20% for glycine,
11.867±0.20% for proline, and 10.51±0.23% for hydroxyproline.
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