摘要
人尿经吸附剂处理，硫酸铵沉淀后，得到胰蛋白酶抑制剂（ＨＵＴＩ）粗品。粗品经ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析及ｔｒｙｐｓｉｎ－Ｓｅｐｈａｒｏｓｅ亲和层析，得到两种ＨＵＴＩ：ＨＵＴＩ１与ＨＵＴＩ２。ＨＵＴＩ１的比活为３５５６ｕｎｉｔｓ／ｍｇ，活性回收率为３５％，含分子量６７０００与２５０００两种组分；ＨＵＴＩ２的比活为３３８５ｕｎｉｔｓ／ｍｇ，活性回收率为３１％，分子量为６７０００。经二步层析纯化，纯化因子达１１．３，而粗品直接上亲和柱所得半纯品的比活为１７０７ｕｎｉｔｓ／ｍｇ，活性回收率为４２％，纯化因子５．５。
Urinary trypsin inhibitor is present in mammals urine. It specially inhibits the activity of trypsin, chymotrypsin and fibrinolysin et al. Human urinary trypsin inhibitor (HUTI) is obtained from normal male urine and has been used in clinic as anticoagulant and antifibrinolysin. Because HUTI is a protein from human, it has no antigenicity and safe in application. We collected fresh urine and treated it with Celite, then eluted Celite with PH 11, 2% ammonia, Crude HUTI was precipitated by 60% (NH 4) 2SO 4. Following purification precedure were chromatography on DEAE-Sephadex A-50 and trypsin-Sepharose: (1) DEAE-Sephadex A-50 Crude HUTI was applied to column of DEAE-Sephadex A-50. The column was eluted with PH 7.8, 0.02M Tris-HCI+0.3M NaCI, PH7.8,0.02M Tris-HCI+0.5M NaCI, PH 7.8,0.02M Tris-HCI+0.8M NaCI. The fraction possessing activity was cllected and lyophilized. (2) Affinity chromatography The semipurified samples from step (1) were applied to column of trypsin-Sepharose respectively. The column was washed by PH 8.0,0.1M NaHCO 3+0.5M NaCI, then eluted by PH 2.5, 0.1M HCOOH+0.5M NaCI, and the adsorbed imhibitor was obtained. From step (1) and (2), two forms of HUTI (HUTI 1 and HUTI 2) were obtained. The specific activity of HUTI 1 was 3356 units/mg with a recovery of 35%, HUTI 2: 3385 units/mg, 31%. The purification factor was 11.3 as compared to that of crude HUTI. HUTI 1 migrated as two bands corresponding to molecular weight of 67000 and 25000 in SDS polyacrylamide gel electrophoresis. HUTI 2 was homogenous and the molecular weight was 67000. As crude HUTI was purified directly on trypsin-Sepharose, the purification factor was 5.5. In experiment, we referred to the epichlorohydrin method to active Sepharose-4B and established the conditions suitable for expending production.
出处
《南京大学学报（自然科学版）》
CAS
CSCD
1997年第1期62-66,共5页
Journal of Nanjing University（Natural Science）
