STRUCTURAL STUDIES ON CARTILAGE COLLAGEN EMPLOYING LIMITED CLEAVAGE AND SOLUBILIZATION WITH PEPSIN
STRUCTURAL STUDIES ON CARTILAGE COLLAGEN EMPLOYING LIMITED CLEAVAGE AND SOLUBILIZATION WITH PEPSIN
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DOI：
10.1021/bi00776a005
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发表时间：
1972-01-01
期刊：
影响因子：
2.9
通讯作者：
MILLER, EJ
中科院分区：
文献类型：
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作者：
MILLER, EJ
胶原蛋白的制备材料与方法。本研究采用10周龄鸡胸骨软骨为起始材料制备胶原蛋白。为此，将组织仔细剥离软骨膜并切成小片，并在大约5体积1.0 m NaCl （pH 7.5, 0.05 m Tris）中连续5天，每天在4下不断搅拌，从新鲜软骨片中提取大部分蛋白多糖成分。按照这些提取程序，切片在蒸馏水中彻底冲洗4，用0.5 m醋酸提取，采用与上述中性盐溶剂相同的程序。酸提后，5000g离心收集切片，再悬浮于0.5 m醋酸中冻干。由此获得的材料在操作上被定义为不溶性软骨胶原。用胃蛋白酶溶解。将1g冻干的软骨切片悬浮在100ml含有100mg胃蛋白酶（PM, Worthington Biochemical Corp.）的0.5 m醋酸中，并将悬浮液在4下持续搅拌18小时，常规溶解软骨胶原。孵育后，将黏性消化混合物在5万g下4℃离心澄清1小时，在澄清后的溶液中加入浓度为0.9 m的结晶NaCl沉淀胶原蛋白。得到的沉淀物在4℃下50000 g离心1小时，再溶解在1.0 m NaCl （pH 7.5, 0.05 m Tris）中。然后将胶原蛋白溶液在大体积0.5 m的醋酸中透析，并按照先前描述的酸溶性骨性胶原蛋白进行进一步纯化（Miller et al., 1967）。胃蛋白酶溶解软骨胶原的层析。在本研究中使用的色谱过程中，除使用DB-GT分光光度计（Beckman instruments, Inc.）进行色谱柱监测外，如前所述（Miller et al., 1969）对柱出水进行监测、记录和收集。对变性后的胃蛋白酶溶解胶原进行羧甲基纤维素（CM-cellulose）层析
Materials and MethodsPreparation of Collagen. The collagen employed as the starting material in the present studies was prepared from the sternal cartilages of 10-week-old chickens. For this purpose, the tissues were carefully stripped of perichondrium and diced into small slices, and the majority of the proteoglycan com-ponents were removed from the fresh cartilage slices by daily extraction with constant stirring at 4 in approximately five volumes of 1.0 m NaCl (pH 7.5, 0.05 m Tris) for 5 successive days. Following these extraction procedures, the slices were thoroughly rinsedat 4 in distilled water and extracted with 0.5 m acetic acid employing the same protocol as outlined above for the neutral salt solvent. Subsequent to acid extrac-tion, the slices were collected by centrifugation at 5000g, re-suspended in 0.5 m acetic acid and lyophilized. The material thus obtained was operationally defined as insoluble cartilage collagen.Solubilization with Pepsin. Cartilage collagen was routinely solubilized by suspending 1 g of lyophilized cartilage slices in 100 ml of 0.5 m acetic acid containing 100 mg of pepsin (PM, Worthington Biochemical Corp.) and incubating the suspen-sion with constant stirring at 4 for 18 hr. Following incubation, the viscous digestion mixture was clarified by centrifuga-tion at 50,000 g for 1 hr at 4, and collagen was precipitated from the clarified solution bythe addition of crystalline NaCl to a concentration of 0.9 m. The resulting precipitate was retrieved by centrifugation at 50,000 g for 1 hr at 4 and redissolved in 1.0 m NaCl (pH 7.5, 0.05 m Tris). The collagen solu-tion was then dialyzed against a large volume of 0.5 m acetic acid at 4 and further purification was achieved as previously described for acid-solublebone collagen (Miller et al., 1967). Chromatography of Pepsin-Solubilized Cartilage Collagen. During the chromatographic procedures used in the present study column effluents were monitored, recorded, and collected as described previously (Miller et al., 1969) with the exception that DB-GT spectrophotometers (Beckman In-struments, Inc.) were employed in column monitoring. Carboxymethyl cellulose (CM-cellulose) chromatography of the pepsin-solubilized collagen after denaturation was performed