Hare E R, Verpoorte J A
Int J Biochem. 1985;17(5):589-95. doi: 10.1016/0020-711x(85)90290-3.
A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography. The results indicate that another protein component binds to the enzyme at pH 8.0. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate. A mol. wt of 40,100 +/- 1800 has been calculated from gel electrophoresis experiments. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.
本文描述了一种从人全唾液中纯化激肽释放酶的快速简便方法。该酶已被纯化约2700倍，产率约为30%。该方法基于通过离子交换色谱法对唾液进行即时分级分离。随后是亲和色谱和高效液相色谱的结合。结果表明，在pH 8.0时，另一种蛋白质成分与该酶结合。在有无十二烷基硫酸钠的情况下，通过凝胶电泳证明了该酶的均一性。根据凝胶电泳实验计算出的分子量为40100±1800。分析超速离心机中的沉降平衡给出的分子量为39700。已测定了氨基酸组成，证实该酶的等电点较低。通过吸收光谱和荧光光谱证明了色氨酸的存在。
