Fujikawa K, Walsh K A, Davie E W
Biochemistry. 1977 May 17;16(10):2270-8. doi: 10.1021/bi00629a036.
Factor XII was purified approximately 14 000-fold from bovine plasma by ammonium sulfate fractionation followed by heparin-agarose, DEAE-Sephadex, CM-cellulose, arginine-agarose, and benzamidine-agarose column chromatography. By this method, about 15 mg of protein was purified from 15 L of plasma with an overall yield of 18%. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal analysis. Bovine factor XII is a glycoprotein with a mol wt of 74 000 as determined by sedimentation equilibrium centrifugation. It contains 13.5% carbohydrate including 3.4% hexose, 4.7% N-acetylhexosamine, and 5.4% N-acetylneuraminic acid. Factor XII is a single polypeptide chain with an NH2-terminal sequence of Thr-Pro-Pro-Trp-Lys-Gly-Pro-?-Lys-His. This sequence is homologous to the reactive-site regions of a number of protease inhibitors. The amino acid sequence of a carboxyl-terminal fragments prepared by cyanogen bromide digestion was found to be Leu-Cys-Ala-Gly-Phe-Leu-Glu-Gly-Gly-Thr-Asp-Ala-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro-Leu-Val-Cys-Glu-Asp-Glu. This sequence is homologous with the active site of a number of plasma serine proteases including thrombin, factor IXa, factor Xa, and plasmin. These data indicate that bovine factor XII is a precursor to a serine enzyme with an inhibitor sequence and a catalytic site located in the same single polypeptide chain.
通过硫酸铵分级沉淀，随后进行肝素琼脂糖、二乙氨基乙基葡聚糖（DEAE - Sephadex）、羧甲基纤维素（CM - cellulose）、精氨酸琼脂糖和苯甲脒琼脂糖柱层析，从牛血浆中纯化出约14000倍的凝血因子XII。通过这种方法，从15升血浆中纯化出约15毫克蛋白质，总产率为18%。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和氨基末端分析判断，纯化后的蛋白质是均一的。通过沉降平衡离心法测定，牛凝血因子XII是一种分子量为74000的糖蛋白。它含有13.5%的碳水化合物，包括3.4%的己糖、4.7%的N - 乙酰己糖胺和5.4%的N - 乙酰神经氨酸。凝血因子XII是一条单一的多肽链，其氨基末端序列为苏氨酸 - 脯氨酸 - 脯氨酸 - 色氨酸 - 赖氨酸 - 甘氨酸 - 脯氨酸 - ？ - 赖氨酸 - 组氨酸。该序列与许多蛋白酶抑制剂的活性位点区域同源。发现通过溴化氰消化制备的羧基末端片段的氨基酸序列为亮氨酸 - 半胱氨酸 - 丙氨酸 - 甘氨酸 - 苯丙氨酸 - 亮氨酸 - 谷氨酸 - 甘氨酸 - 甘氨酸 - 苏氨酸 - 天冬氨酸 - 丙氨酸 - 半胱氨酸 - 谷氨酰胺 - 甘氨酸 - 天冬氨酸 - 丝氨酸 - 甘氨酸 - 甘氨酸 - 脯氨酸 - 亮氨酸 - 缬氨酸 - 半胱氨酸 - 谷氨酸 - 天冬氨酸 - 谷氨酸。该序列与许多血浆丝氨酸蛋白酶的活性位点同源，包括凝血酶、因子IXa、因子Xa和纤溶酶。这些数据表明，牛凝血因子XII是一种丝氨酸酶的前体，其抑制剂序列和催化位点位于同一条单一的多肽链中。
