河蚌C反应蛋白一级结构分析

Primary Structure of Anodonta C-reactive Protien

经亲和层析纯化的河蚌 C反应蛋白 ( CRP)具有 SDS- PAGE纯度 ,用经改进的双偶联 Ed-man方法测得其 N端残基为谷氨酸 ,而不是高等动物 (人与家兔 ) C反应蛋白 N端的焦谷氨酸 .河蚌 C反应蛋白 N端的一级结构由固相 Edman方法测得 ,依次为 H2 N- E- T- A- Y- S- C- I- T- A- V- ;C端的一级结构由羧肽酶 A降解法测得 ,依次为 - L/V- S- S- T- Y- COOH,也不同于人和家兔的 C反应蛋白 .在河蚌 CRP的胰蛋白酶酶解肽段中 ,其 N端及 C端的结构也得到了证实 .河蚌 C反应蛋白经 V8蛋白内切酶酶解 ,溴化氰裂解 ,肽段经 HPLC反相柱分离 ,共得到 35个肽段 ,所有肽段的氨基酸序列均由气相氨基酸自动分析仪测得 .结合河蚌 C反应蛋白的胰蛋白酶酶解肽段的分析结果 ,其一级结构已初步拼接完成 .在其一级结构中发现有类似于其它 CRP的 Ca2 + 结合部位和磷酸胆碱结合部位 .河蚌 C反应蛋白的分子结构中存在微观不均一性 .从已知河蚌 C反应蛋白的分子特点 ,包括分子量 ,糖基化比例 ,一级结构不均一等特点 ,可以推测它与高等动物的免疫蛋白有许多相关之处 .对于河蚌 C反应蛋白分子结构的分析 ,将有助于免疫系统蛋白的发生 。

Anodonta C reactive protein (CRP), a primeval CRP, was purified from the body fluid of Anodonta Woodiana by affinity chromatography. Purified Anodonta CRP showed a single band in SDS PAGE. Contrasted with the pyroglutamic acid that was found in human or rabbit CRP, the N terminal residue of the Anodonta CRP was Glu. The sequence of the first ten amino acids at the N terminal of the Anodonta CRP was H 2N E T A Y S C I T A V ,which was determined using the modified Edman degradation. The C terminal sequence of the Anodonta CRP had been determined by the carboxypeptidase A degradation. It was L/V S S T Y COOH, different from any other CRP obtained from different species (human or rabbit). The N and C terminal sequences were also found and proved in the tryptic peptides sequences. This Anodonta CRP was digested with trypsin, V8 protease and CNBr respectively. The peptides were separated and collected with the C 18 reversed phase column HPLC and all of the 35 peptides from the HPLC or Sephacryl 200 gel filtration were sequenced. The major structure of the Anodonta CRP was determined by splicing of the sequences from the tryptic peptides, V8 peptides, and CNBr peptides. Both Ca 2+ binding and phosphorylcholine (PC) binding sequences were found in the primary structure. However, except for the two binding sites there was no similar structure to be found between Anodonta CRP and CRP from other species. Additionally, variability was found in the primary sequence of the Anodonta CRP. This phenomenon has been found in the limulus CRP and suggests that the Anodonta CRP is coded by multiple genes.