ADSORBENTS FOR AFFINITY CHROMATOGRAPHY - USE OF N-HYDROXYSUCCINIMIDE ESTERS OF AGAROSE
ADSORBENTS FOR AFFINITY CHROMATOGRAPHY - USE OF N-HYDROXYSUCCINIMIDE ESTERS OF AGAROSE
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DOI：
10.1021/bi00762a013
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发表时间：
1972-01-01
期刊：
影响因子：
2.9
通讯作者：
PARIKH, I
中科院分区：
文献类型：
--
作者：
CUATRECASAS, P;PARIKH, I
佩德罗·库特雷卡斯*·！和Indu Parikh摘要：描述了制备琥珀酸氨基烷基琼脂糖衍生物的W-羟基琥珀酰亚胺酯的方法。这些琼脂糖的活性酯衍生物在二恶烷中储存数月都是稳定的。详细研究了琼脂糖的活性酯与各种氨基酸和胰酶的反应。在pH为6-9的范围内，与未质子化形式的伯族脂肪族或芳香族氨基形成稳定的酰胺键非常迅速(在4)。在所测试的氨基酸官能团中，只有巯基有效地与游离氨基竞争反应。已描述了用于蛋白质亲和层析的选择性琼脂糖吸附剂(Cuatrecasas等人，1968；Porath等人，1967；Cuatrecasas，1970,1972；Cuatrecasas和Anfinsen，1971a，b)的各种步骤的IV-羟基琥珀酰亚胺酯衍生物。有大量证据表明，酶的成功纯化往往取决于使用一种吸附剂，其中配体与基质主链在空间上分离(Cuatrecasas，1972)。这通常是通过将配体连接到琼脂糖衍生物上实现的最好的方法，琼脂糖衍生物包含以功能部分终止的长碳氢化合物延伸，例如伯氨基或来自约翰霍普金斯大学医学院的医学部和药理学和实验治疗学部的CAR-F，巴尔的摩，马里兰州21205。1972年1月31日收到。由国家关节炎和代谢性疾病研究所资助的AM14956赠款。美国公共卫生服务研究职业发展奖AM31464的获得者。琼脂糖可以通过非常简单和温和的步骤将蛋白质和含有配体的复杂氨基固定到琼脂糖上。通过长的碳氢化合物延伸将偶联产物与基质主干分开。类似的程序也可以用来将配体和蛋白质连接到多孔玻璃微珠上。此外，这些研究还表明，琼脂糖珠对某些有机溶剂，如二恶烷和甲醇有很好的耐受性。活化的琼脂糖衍生物经冷冻干燥去除二恶烷后，呈粉末状很稳定，悬浮在水介质中很容易膨胀并与氨基反应。羧基。人们利用各种化学反应将配体与这种“长臂”琼脂糖衍生物偶联(Cuatrecasas，1972)。然而，如果不使用复杂的封闭和解封这些基团中的一个或多个的程序，就没有普遍适用的方法可用于偶联配体，例如含有一个以上官能团的氨基酸。此外，到目前为止，如果不通过蛋白质的酪氨基或组氨酸残基偶联，或者不引入分子间蛋白质的交联和聚合，就很难将蛋白质共价连接到任何含有官能团(如氨基、羧基、重氮离子、溴乙酰基)的固体载体上。本报告详细介绍了长臂琼脂糖衍生物的活性羧酸酯的制备和用途，这些活性羧酸酯在无水介质中可以长期稳定。在温和的条件下，含有游离氨基的配体和蛋白质可以通过酰胺键与这些活性衍生物快速偶联(《水生物化学》第11卷，第1期，第197-22291期)。
Pedro Cuatrecasas*·! and Indu Parikh abstract: Procedures are described for the preparation of the W-hydroxysuccimmide ester of succinylated aminoalkyl agarose derivatives. These active ester derivatives of agarose are stable for months when stored in dioxane. The reaction of the active ester of agarose with various amino acids and with trypsin has been studied in detail. Stable amide linkages are formed very rapidly (at 4) with the unprotonated form of primary aliphatic or aromatic amino groups in the pH range of 6-9. Of the amino acid functional groups tested, only sulfhy-dryl groups compete effectively with free amino groups for reaction. The iV-hydroxysuccinimide ester derivative of vV arious procedures have been described for the prepara-tion of selective agarose adsorbents for use in affinity chromatography of proteins (Cuatrecasas et al., 1968; Porath et al., 1967; Cuatrecasas, 1970, 1972; Cuatrecasas and Anfinsen, 1971a, b). There is abundant evidence that successful purifica-tion of enzymes frequently depends on utilizing an adsorbent in which the ligand is spatially separated from the matrix backbone (reviewed by Cuatrecasas, 1972). This is generally best accomplished by linking the ligand to agarose derivatives which contain lengthy hydrocarbon extensions terminating in a functional moiety such as a primary amino group or a car-f From the Department of Medicine and the Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Received January 31, 1972. Supported by Grant AM14956 from the National Institute of Arthritis and Metabolic Diseases. t Recipient of U. S. Public Health Service Research Career Develop-ment Award AM31464. agarose can be used byvery simple and mild procedures to immobilize proteins and complex amino group containing ligands to agarose. The coupled products are separated from the matrix backbone by lengthy hydrocarbon extensions. Similar procedures can be used toattach ligands and proteins to porous glass beads. These studies in addition demonstrate that agarose beads tolerate quite well certain organic solvents such as dioxane and methanol. The activated agarose deriva-tives, after lyophilization to remove dioxane, are quite stable in the powder form and readily swell and react with amino groups when suspended in aqueous medium. boxyl group. A variety of chemical reactions have been used to couple ligands to such “long-armed” agarose derivatives (Cuatrecasas, 1972). However, no generally applicable pro-cedure is available for coupling ligands such as amino acids which contain more than one functional group without using complicated blocking and deblocking procedures of one or more of these groups. Furthermore, it has heretofore been very difficult to covalently attach proteins to any solid support which contains functional groups (eg, amino, carboxyl, di-azonium, bromoacetyl) without coupling through the tyrosyl or histidyl residues of the protein or without introducing inter-molecular protein cross-links and polymerization. This report describes in detail the preparation and use of active carboxylic acid esters of long-armed agarose derivatives which are stable in anhydrous media over prolonged periods of time. Ligands and proteins which contain free amino groups can be coupled very rapidly through amide linkage to these activated derivatives under mild conditions (aqueous biochemistry, vol. 11, no. 1 2, 1 9 7 2 2291