Yu Q, Kandegedara A, Xu Y, Rorabacher D B
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Anal Biochem. 1997 Nov 1;253(1):50-6. doi: 10.1006/abio.1997.2349.
Of the 20 well-known buffers proposed by Good, all but 3 form metal ion complexes which can result in serious interferences, particularly in protein analyses. The structural features responsible for such complex formation have been identified. Based on a mechanistic analysis of the metal complexation process, it is proposed that tertiary amine compounds, having N-substituents which are ethyl or larger, are sterically inaccessible for initial bond formation with solvated metal ions in aqueous solution. Thus, in the absence of donor atoms on the alpha-, beta-, or gamma-carbons, metal complexation cannot proceed. The proposed noncomplexing compounds include Good's 3 noncomplexing buffers (Mes, Mops, Pipes) plus six related species. Mixed-mode acid dissociation constants (hydrogen ion in terms of activity, conjugate acid-base species in molar concentrations) have been determined for all compounds in their protonated form at 25 degrees C, mu = 0.10 M (NaNO3). The values for four compounds in this series are reported here for the first time: viz., N,N'-diethylpiperazine for which log Ka1m = 4.67 +/- 0. 03 and log Ka2m = 8.83 +/- 0.02; N,N,N',N'-tetraethylmethylenediamine for which log Ka1m < 1 and log Ka2m = 11.01 +/- 0.03; N,N'-diethyl-N,N'-bis(3-sulfopropyl)ethylenediamine for which log Ka1m = 5.75 +/- 0.03 and log Ka2m = 9.37 +/- 0.02; and piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) for which log Ka1m = 2.81 +/- 0.01 (the value of log Ka2m having been previously reported).
在古德提出的20种著名缓冲剂中,除3种外,其余缓冲剂都会形成金属离子络合物,这可能导致严重干扰,尤其是在蛋白质分析中。已确定了导致此类络合物形成的结构特征。基于对金属络合过程的机理分析,有人提出,具有乙基或更大N-取代基的叔胺化合物在水溶液中与溶剂化金属离子形成初始键时,空间位阻较大,难以接近。因此,在α-、β-或γ-碳上没有供体原子的情况下,金属络合无法进行。提议的非络合化合物包括古德的3种非络合缓冲剂(MES、MOPS、PIPES)以及六种相关物质。已测定了所有质子化形式的化合物在25℃、μ = 0.10 M(硝酸钠)条件下的混合模式酸解离常数(以活度表示氢离子,以摩尔浓度表示共轭酸碱物种)。本系列中四种化合物的值首次在此报告:即N,N'-二乙基哌嗪,其log Ka1m = 4.67±0.03,log Ka2m = 8.83±0.02;N,N,N',N'-四乙基甲二胺,其log Ka1m < 1,log Ka2m = 11.01±0.03;N,N'-二乙基-N,N'-双(3-磺丙基)乙二胺,其log Ka1m = 5.75±0.03,log Ka = 9.37±0.02;以及哌嗪-N,N'-双(2-乙磺酸)(PIPES),其log Ka1m = 2.81±0.01(log Ka2m的值此前已报告)。