Biochemical Studies on Human Inter- trypsin Inhibitor

胰蛋白酶间抑制剂（ITI）是存在于人血浆中的七种丝氨酸蛋白酶抑制剂之一。本研究建立了一种实用的分离ITI的方法，并尝试确定ITI的多肽链结构。通过Q-Sepharose、DEAE-Sephacel和肝素-Sepharose层析从人血浆中纯化ITI。用此方法从1 L人血浆中可获得约50 mg ITI。2）ITI的多肽链结构。还原和非还原ITI在SDS-聚丙烯酰胺凝胶电泳上得到210 kDa的单一条带，表明ITI由单一多肽链组成。然而，单链ITI给出两个N-末端氨基酸残基：Ser-Leu-Pro-和Ala-Val-Leu-。一些ITI制备物给出140 kDa和70 kDa的额外条带，其通过使用Mono-Q柱的高效液相色谱法分离。140 kDa片段保留了胰蛋白酶抑制活性，其N端为Ala-Val-Leu，而70 kDa片段的N端为Ser-Leu-Pro。这些结果表明ITI由140和70 kDa的两条链组成，它们以独特的连接方式连接。ITI在pH12、37 ℃下孵育60分钟导致ITI裂解成两个70kDa和45kDa片段.发现抑制活性与45 kDa片段相关。用透明质酸酶处理45 kDa片段导致产生具有抑制活性的25 kDa片段。此外，用透明质酸酶处理完整的ITI产生25 kDa的抑制片段和170 kDa的片段。这些结果表明ITI由25、70和100 kDa的三条链组成，并且25 kDa的活性结构域通过20 kDa的碳水化合物链连接到100 kDa的链。ITI的碱处理似乎切断了连接三条多肽链的连接。

Inter- trypsin inhibitor (ITI) is one of seven serine protease inhibitors present in human plasma. In the present study, we have established a practical method for isolation of ITI and attempted to determine polypeptide chain structure of ITI.1) Purification of ITI. ITI was purified from human plasma by chromatographies with Q-Sepharose, DEAE-Sephacel, and heparin-Sepharose. By this method, about 50 mg of ITI were obtained from 1 L human plasma.2) Polypeptide chain structure of ITI. Reduced as well as non-reduced ITI gave a single band on SDS-polyacrylamide gel electrophoresis of 210 kDa, suggesting ITI to be composed of a single polypeptide chain. However, the single chain ITI gave two N-terminal amino acid residues; Ser-Leu-Pro- and Ala-Val-Leu--. Some ITI preparations gave additional bands of 140 kDa and 70 kDa, which were separated by high performance liquid chromatography using a Mono-Q column. The 140 kDa fragment retains the trypsin inhibitory activity and its N-terminal was Ala-Val-Leu, while the N-terminal of 70 kDa fragment was Ser-Leu-Pro. These results suggested that ITI is composed of two chains of 140 and 70 kDa which are-connected with a unique linkage.3) Chemical and enzymatic cleavage of ITI. Incubation of ITI at pH 12 for 60 min at 37 C resulted in the cleavage of ITI into two 70 kDa and 45 kDa fragments. Inhibitory activity was found to be associated with 45 kDa fragment. Treatment of the 45 kDa fragment with hyarulonidase resulted in the production of 25 kDa fragment having inhibitory activity. In addition, treatment of intact ITI with hyarulonidase produced 25 kDa inhibitory fragment and 170 kDa fragment. These results suggested that ITI is composed of three chains of 25,70,and 100 kDa and that the active domain of 25 kDa is connected to via carbohydrate chain of 20 kDa to the 100 kDa chain. Alkalinetreatment of ITI seems to cleave the linkages connecting the three polypeptide chains.