EXCITATION ENERGY TRANSFER AND THE QUANTITATIVE STUDY OF THE ANTIBODY HAPTEN REACTION
EXCITATION ENERGY TRANSFER AND THE QUANTITATIVE STUDY OF THE ANTIBODY HAPTEN REACTION
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DOI：
10.1073/pnas.46.11.1470
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发表时间：
1960-01-01
影响因子：
11.1
通讯作者：
EISEN, HN
中科院分区：
文献类型：
--
作者：
VELICK, SF;PARKER, CW;EISEN, HN
蛋白质的色氨酸荧光在蛋白质与分子的络合物或结合物中被猝灭，这些分子在300到400微米范围内具有吸收。这一现象以前应用于酶辅酶复合体的研究，也适用于含有适当抗原或半抗原的抗体复合体。针对2，4-二硝基苯基(DNP)的抗体的荧光量子产率因[epsilon]-N-DNP-赖氨酸的结合而降低约70%，是络合物形成的灵敏指示剂。结合化学计量比为每摩尔纯化抗体2摩尔半抗原，在7[度]时的解离常数为2.4×10-9[图]。这种大小的离解常数是用其他方法无法接近的。在pH 1.5至11和温度7[度]至60[度]范围内已研究了半抗原结合。络合物形成的[Delta]H[度]和[Delta]F[度]分别为-11.3和-8.6kcal摩尔-1，[Delta]S[度]为9EU。一些同源单价半抗原结合较弱，并与二价半抗原形成线状聚集体。用直接滴定的方法确定纯化抗体经木瓜酶蛋白降解产生的可溶性片段I和II为单价抗体片段，而用Porter‘’S方法制备的结晶片段III不带半抗原结合部位。单价抗体片段可以用荧光法与完整的多价DNP抗原进行定量滴定，因为它们形成了可溶性的络合物，其中抗体和抗原的荧光都被抗原上的DNP配体猝灭。纯化的抗DNP抗体与半抗原相互作用的结合部位具有高度的均一性，但在可逆电泳界铺展中的统计不均一性与非特异性的[γ]-球蛋白具有相同的大小。荧光猝灭法也被应用于研究针对对偶氮苯基砷酸盐衍生物的抗体的半抗原相互作用。在半抗原复合体内的猝灭相互作用中，每个结合的DNP分子捕获了8到9个色氨酸残基的激发能量。抗体的这些色氨酸残基位于不同的区域内，这些区域不重叠，其边界与木瓜酶的蛋白水解点大致重合。
The tryptophane flourescence of proteins is quenched in complexes or conjugates of the proteins with molecules that have absorption in the 300 to 400 mu region. This phenomenon, applied previously to the study of enzyme coenzyme complexes is also applicable to antibody complexes with appropriate antigens or haptens. The fluorescence quantum yield of the antibody directed against the 2,4-dinitrophenyl group (DNP) is diminished about 70% by the binding of [epsilon]-N-DNP-lysine and serves as a sensitive indicator of complex formation. The binding stoichiometry is 2 moles of hapten per mole of purified antibody and the dissociation constant at 7[degree] is 2.4 10-9 [image]. Dissociation constants of this magnitude are not approachable by other methods. Hapten binding has been studied over the pH range 1.5 to 11 and over the temperature range 7[degree] to 60[degree]. [DELTA]H[degree]and [DELTA]F[degree]for complex formation are -11.3 and -8.6 kcal mole-1 and [DELTA]s[degree]is 9 eu. Weaker binding of some homologous univalent haptens and the formation of linear aggregates with a divalent hapten are described. It is established by direct titration that the soluble fragments I and II produced by papain proteolysis of the purified antibody, are univalent antibody pieces and that the crystalline fragment III, prepared by Porter''s method, carries no hapten binding site. The univalent antibody fragments may be titrated quantitatively, by the fluorometric method, with intact multivalent DNP antigen since they form soluble complexes in which the flourescence both of the antibody and antigen is quenched by the DNP ligands on the antigen. The binding sites of the purified anti-DNP antibody exhibit a high degree of homogeneity in their interactions with haptens but the statistical inhomogeneities, in reversible electrophoretic boundary spreading are of the same magnitude as those of nonspecific [gamma]-globulin. The fluorescence quenching method is also applied to the study of hapten interactions of an antibody directed against derivatives of p-azophenylarsonate. In the quenching interaction within the hapten complex each molecule of bound DNP captures the excitation energy of 8 to 9 tryptophane residues. These groups of tryptophane residues of the antibody lie within separate domains which do not overlap and the boundaries of which coincide roughly with the points of proteolytic cleavage by papain.