（一)囓齒類貯精囊分泌蛋白（ SVS-Ⅳ）的純化及研究 （二)β-雨傘節毒素的酵素免疫分折

Abstract

近年來，對於Rat貯精囊分泌蛋白的研究，已知其含有5個主要蛋白，有關其生物功能，目前尚不清楚。本實驗綜合硫銨?析、離子交換層析及再層析，來純化Mouse SVS，經由SDS-PAGE電泳分析其純度，發現貯精囊的分泌蛋白以SVS-Ⅳ含量最多。由胺基酸分析，其多勝鏈的分子量為10 Kd，含有多量的絲胺酸，缺少色胺酸及半胱胺酸，與Rat SVS-Ⅳ有許多性質相似，比較二者胺基酸次序，發現有60%以上的同源關係。經CD spectropolarimeter分析，2個蛋白均以不規則的二級結構存在。 過去的結果已知鑭系元素可當成Ca+2的替代物，藉此來研究蛋白分子是否具有Ca+2-binding sites。利用螢光分析，發現SVS-Ⅳ307 nm螢光減少及Tb+3 486nm螢光增強的現象，來研究Tb+3與SVS-Ⅳ的結合。在中性溶液中，Tb+3和mSVS-Ⅳ, rSVS-Ⅳ的結合常數分別是(2.45士0.19)x103M-1, (9.4±0.93)x102M-1。改變溶液pH值，Tb+3與SVS-Ⅳ的結合常數並未改變，但SVS-Ⅳ的立體結構可能已發生變化。 由CD光譜的研究可知，Ca+2對SVS-Ⅳ的二級結構沒有影響，且Ca+2也無法取代Tb+3-SVS-Ⅳ之位置，由此推斷SVS-Ⅳ，可能沒有Ca+2鍵結的位置。另外，SVS-Ⅳ經證明為非磷化蛋白，且mSVS-Ⅳ不具有醣分子。 台灣雨傘節(Bungarus multicintus)毒液中之β-Bungaro-toxin是由兩條多勝鏈(A, Bchains)以雙硫鍵連接。抗血清經親和層析純化得到的抗體，可同時與分開的A, B鏈反應，對其他鍵前神經毒素如：Caudoxin、Crotoxin、Mojave toxin、及Taipoxin則無反應，利用此專一性的抗體，綜合Streptavidin-biotin system，發展酵素免疫分析，提高測定的靈敏度。

The secretory protein IV (SVS-IV) was purified from the seminal vesicle of mouse by a combination of ammonium sulfate fractionation, ion exchange chromatography to yield a product that appeared homogeneous in SDS polyacrylamide gel electrophoresis. SVS-IV is the most abundant protein in the seminal vesicle secretions. From the amino acid sequence, the molecular weight is about l0Kd; it lacks tryptophan and cysteine; serine comprises a relatively large proportion of this protein. There are similar properties in rat SVS-IV, too. Furthermore, mouse SVS-IV amino acid sequence has 60% homology with rat SVS-IV. The circular dichroic spectra indicated the absence of ordered secondary structure in the SVS-IV molecule. The interactions of terbium and SVS-IV were investigated by enhancement of Tb(III) fluorescence (λex280nm, λem486nm) and decrease in the tyrosine fluorescence(λex280nm,λem307nm). The association constants of Tb(III) to mouse SVS-IV and to rat SVS-IV were determined to be about (2.45±0.19)x103 M-1 and (9.4±0.93)x102 M-1, respectively. In line with the pH dependence of Tb(III)-SVS-IV complex formation and with the constant of binding affinity as Tb(III) bound to SVS-IV at pH 6.5, 7.0 and 7.5, we suspected that pH-induced conformational change in the protein molecule was accompanied by an increase of the energy transfer efficiency. The stepwise addition of Ca(II) to Tb(III)-SVZ-IV solution didn’t yield a large replacement of tyrosine- sensitized Tb(III) fluorescence enhancement. From the results of terbium emission, we believed that rSVS-IV and mSJS-IV had no Ca++-binding site. Quantitation of phosphate suggested that SVS-IV existed in the non-phosphorylated form before its release from the gland lumen. The mouse SVS-IV antiserum was highly specific, which didn’t show cross-reaction with rat SVS-IV. β-Bungarotoxin, a presynaptically active polypeptide, consists of two subunits which are linked by S-S bridges. We had prepared β-BuTx antibody which bound specifically to either subunit. The antibody gave very low binding to both non-neurotoxin phospholipase A2 and other presynaptic neurotoxins such as Caudoxin, Crotoxin, Mojave toxin and Taipoxin. Using the antibody, we had developed an enzyme immunoassay which could detect β-BuTx to an amount as low as 15 pg.