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Atelocollagen separation and characterization
Konečná, Zuzana ; Zubal, Lukáš (referee) ; Vojtová, Lucy (advisor)
The aim of the presented bachelor thesis was to optimize separation of soluble atelocollagen out of commercially available bovine collagen and its characterization. Collagen went through an enzymatic digestion using pepsin as a protease and subsequently purified by filtration, centrifugation and dialysis. Obtained samples were characterized using infrared spectrometry, polyacrylamide gel electrophoresis and Hartree-Lowry assay for total protein determination. Suitable digestion time range to gain sufficient yields was decided from 60 – 70 hours at pepsin/collagen weight ratio 1:25. Highest yield (72.6 %) was obtained at mentioned ratio after 66 hours of reaction. Sufficient filtration, which was decisive for final product purity, was assured by combination of several filtration techniques with centrifugation. Quality of prepared product according to their molecular weight was characterized by gel electrophoresis. Solubility and its dependence on pH and NaCl concentration in solution were measured by Hartree – Lowry assay. Ratio between helical collagen and denaturated collagen was measured by infrared spectroscopy. Obtained spectra yet did not seem fully reliable and provided only a rough estimate. More accurate information about structure could be achieved by using circular dichroism spectroscopy.
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Atelocollagen separation and characterization
Konečná, Zuzana ; Zubal, Lukáš (referee) ; Vojtová, Lucy (advisor)
The aim of the presented bachelor thesis was to optimize separation of soluble atelocollagen out of commercially available bovine collagen and its characterization. Collagen went through an enzymatic digestion using pepsin as a protease and subsequently purified by filtration, centrifugation and dialysis. Obtained samples were characterized using infrared spectrometry, polyacrylamide gel electrophoresis and Hartree-Lowry assay for total protein determination. Suitable digestion time range to gain sufficient yields was decided from 60 – 70 hours at pepsin/collagen weight ratio 1:25. Highest yield (72.6 %) was obtained at mentioned ratio after 66 hours of reaction. Sufficient filtration, which was decisive for final product purity, was assured by combination of several filtration techniques with centrifugation. Quality of prepared product according to their molecular weight was characterized by gel electrophoresis. Solubility and its dependence on pH and NaCl concentration in solution were measured by Hartree – Lowry assay. Ratio between helical collagen and denaturated collagen was measured by infrared spectroscopy. Obtained spectra yet did not seem fully reliable and provided only a rough estimate. More accurate information about structure could be achieved by using circular dichroism spectroscopy.
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