- 作者: 羅銅壁; 張文章; 張陳賢
- 作者服務機構: 國立台灣大學化學研究所; 中央研究院生物化學研究所
- 中文摘要: (一))溶血素胺基酸排列次序之研究- 第三部 溶血素先用還原劑DTT或DTE予以還原,然後用lodoacetic Acid烷化。由此得到的衍生物再用Malefic Anhydride處理使Lysine之(-胺基受到修飾。然後用Trypsin水解以得限定 tryptic peptides 這些 tryptic peptides 再用各種方法分離和純化。另外由CNBr裂解所得較大peptide, CB-I用 JEOL JAS-47K胺基酸序列自動分析儀做Edman degradation,溶血素的部分胺基酸排列次序推測如下: Asn. Leu. Tyr. Phe. Gln Lys. Asn. Met. Ile. Glx. X. Thr. Val.Ala Pro. X. Ala. Arg. Trp. Ala. Asp. Phe. Ala. Ala. Thr. Thr...(二)飯匙倩蛇毒中微量毒素成份之研究 台灤產飯匙倩粗毒以CM-cellulose柱層分析怯分離出八個成分,在第八成分(心臟毒素)之後常伴隨一微量成分之小?(peak)。經多次收集此成分,我們再利用CM-cellulose柱層分析重新分離,經兩次的再分離,終於得到一均質的蛋白質(Fr-9-II)此蛋白質經polyacrylamide膠體電動泳動法及N-Terminal分析均證明其為一均勻的純質。 由膠體柱層分析法(gel ftltration)測定分子量為5200 ~5600,胺基酸分析測定,知道含有46~50個胺基酸,含有兩個雙硫鍵,且不含自由硫氫基(free sulfhydryl group)。 初步的生物試驗,顯示毒性試驗濃度達100 μg'g mice仍不能使實驗的小鼠致死,當加此蛋白質溶液於小雞的頸項肌,並無顯著的肌肉收縮作用,但作Anti-cholinesterase活性測定,卻顯示較粗毒及心臟毒素(Fr 8)活性為大,此種活性之意義有待進一步之探討。(三)Bungarus fasciatus蛇毒化學成份之分離研究 Bungarus fasciatus蛇毒經CM-cellulose柱層分析在醋酸銨緩衝溶液中以twostage gradient elution分離得到13個fractions。就其每一fraction,分別做毒性試驗及酵素活性之檢定及分佈。以acrylamide gel電泳法證明 CM-cellulose柱層分析自粗蛇毒分離出之fractious尚非均質。 Fr V及F'r VI含有心臟毒活性,會引起骨骼肌之contracture:;而毒素最強之Fr VIII則有神經毒作用,能產生神經肌之blockade。 F'r VI經DEAF-cellulose及Sephadex G-50柱層分析作進一步純後,可得一均質毒素(Fr-VI-A-I)。此精製毒素含有胺基酸數目84~87個,其多胜鏈並以四對雙硫鍵作分子內鍵結,以分子過爐法測得分子量為9,000 ~ 10,500,若以84~87個胺基酸數目計算則分子量為9,300~9,700。 本蛇毒進一步研究正在進行中。
- 英文摘要: 1. Studies on the Amino Acid Sequence of Phospholipase A-Part 3 (Prepared in collaboration with Mr. Chang Wen-Chang, instructor) Phospholipase A was treated with reducing agents, DTT (dithiothritol) or DTE (dithioerythritol) in different media and followed by alkylation with iodoacetic acid. The S-carboxymethylated Phospholipase A was then male- ylated with maleic anhydride to block the ε-amino groups of lysine residues. Then it was digested with trypsin to give restrictive tryptic peptides. These tryptic peptides were fractionated and purified. The larger peptide from cyanogen bromide cleavage, CBI, was subjected to Edman degradation using JEOL JAS47-K Sequence Analyzer. The tentative partial amino acid seque- nce of phospholipase A was deduced as follows: Asn-Leu-Tyr-Phe-Gln-Lys-Asn-Met-Ile-Glx-X-Thr-Val-Ala Pro-X-Ala-Arg- Trp-Ala-Asp-Phe-Ala-Ala-Thr-Thr2. The Isolation and Characterization of a Minor Toxic Component from Formosan Snake (Naja naja atra) Venom (Prepared in collaboration which Mr. Chang Chen-Shying, assistant) The fractionation of Formosan snake venom by CM-cellulose chromatographygave eight fractions accompanied by a small fraction (Fr 9) following the fraction-8 (cardiotoxin). After collecting enough amounts of this minor fraction, the re-chromatography by CM-cellulose chromatography was followed and a homogeneousprotein fraction (F-9-II) was obtained. Its purity was confirmed by polyacrylamidegel electrophoresis and N-terminal analysis. The molecular weight was determined to be 5200-5600 by gel-filtration. It iscomposed of 46-50 amino acid residues determined by amino acid analyzer and itconsists of 2 disulfide bonds without free sulfhydryl group. The preliminary biological test showed that the concentration up to 100 μg/gRf mice did not lead to the death of experimental mice and np obvious contracture Tung-Bih Lo, Wen-Chang Chang, Chen-Shving Changeffect when applied to the chick biventer cervicis muscle, but with greater anti-cholinesterase activity than the crude venom and cardiotoxin (Fr-8). The signific-ance of this anti-cholinesterase activity is under further study.3. Studies on Isolation of Bungarus fasciatus Venom The venom of Bungarus fasciatus was separated on a CM-cellulose column intothirteen fractions by two stage gradient elution with ammonium acetate buffer. Thetoxicity and activity distribution of some enzymes for each fraction has been in-vestigated. All of the fractions obtained from CM-cellulose column chromatographyare heterogeneous as monitored by acrylamide gel electrophoresis. Fr V and Fr VI showed cardiotoxic activity and Fr VIII, the most toxic frac-tion, showed neurotoxic action, which cause skeletal muscle contracture and neu-romuscular blockade, respectively. By chromatography of Fr VI on DEAE-cellulosecolumn and then on a column of Sephadex G-50 for further purification, a them-ically homogeneous toxin (Fr VI-A-I) was obtained. This purified protein consist-ed of a polypeptide chain of 84 to 87 amino acid residues cross-linked by fourdisulfide bridges. Its molecular weight was 9,000-10,500 or 9,300-9,700 as estimatedeither by analytical gel filtration or amino acid analysis, respectively. Further studies of this venom are in progress.
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