A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: A surface plasmon resonance study
A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: A surface plasmon resonance study
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DOI：
10.1021/ac980959t
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发表时间：
1999-02-15
影响因子：
7.4
通讯作者：
Whitesides, GM
中科院分区：
文献类型：
--
作者：
Lahiri, J;Isaacs, L;Whitesides, GM
本文描述了将10种蛋白质和2种低分子量配体固定在金链烷硫酸酯混合自组装单层（sam）上，由高（乙二醇）端硫醇1 (HS(CH2)(11)(OCH2CH2)(3)OH) （chi(1) = 1.0-0.0）和长(羧酸端硫醇2 (HS(CH2)(11)(OCH2CH2)(6)OCH2CO2H) （chi(2)= 0.0-1.0）生成。固定是通过两个步骤实现的：从SAM中2的羧基生成反应性n -羟基琥珀酰亚胺酯，并将这些基团与蛋白质或配体上的胺偶联。由于该方法涉及一种容易制备的常见反应中间体，因此它提供了一种方便的方法，将配体附着在使用表面等离子体共振光谱的SAMs上进行研究（原则上，其他生物分析方法使用衍生化的SAMs在金、银和其他表面上）。偶联缓冲液的pH值、蛋白质的浓度、蛋白质溶液的离子强度和蛋白质的分子量都影响被固定的蛋白质的数量。对于详细研究的蛋白质-碳酸酐酶和溶菌酶-当使用低离子强度溶液，pH值约为蛋白质等电点（pl）以下一个单位，浓度约为0.5 mg mL（-1）时，固定化蛋白质的数量最高。通过比较碳酸酐酶和万古霉素分别与固定苯磺酰胺和n - α - ac - lys -d - ala -d - ala基团结合的动力学和热力学常数，在混合SAMs上（通过本文描述的方法）和在市售底物的羧甲基右旋糖酐基质上得到（对于这些系统）k -d、k（off）和k（on）基本上无法区分的值。
This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed self-assembled monolayers (SAMs) of alkanethiolates on gold generated from the hi(ethylene glycol)-terminated thiol 1 (HS(CH2)(11)(OCH2CH2)(3)OH) (chi(1) = 1.0-0.0) and the longer, carboxylic acid-terminated thiol 2 (HS(CH2)(11)(OCH2CH2)(6)OCH2CO2H) (chi(2)= 0.0-1.0). The immobilization was achieved by a two-step procedure: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups of 2 in the SAM and coupling of these groups with amines on the protein or Ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs for studies using surface plasmon resonance spectroscopy (and, in principle, other bioanalytical methods that use derivatized SAMs on gold, silver, and other surfaces). These SAMs were resistant to non-specific adsorption of proteins having a wide range of molecular weights and isoelectric points, The pH of the coupling buffer, the concentration of protein, the ionic strength of the solution of protein, and the molecular weight of the protein all influenced the amount of the protein that was immobilized, For the proteins investigated in detail-carbonic anhydrase and lysozyme-the highest quantities of immobilized proteins were obtained when using a low ionic strength solution at a value of pH approximately one unit below the isoelectric point (pl) of the protein, at a concentration of similar to 0.5 mg mL(-1). Comparisons of the kinetic and thermodynamic constants describing binding of carbonic anhydrase and vancomycin to immobilized benzenesulfonamide and N-alpha-Ac-Lys-D-Ala-D-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially available substrates yielded (for these systems) essentially indistinguishable values of K-d, k(off), and k(on).