Xie Yong, Jiang Yanan, Ben-Amotz Dor
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Anal Biochem. 2005 Aug 15;343(2):223-30. doi: 10.1016/j.ab.2005.05.038.
Raman spectra of phosphorylated amino acids and peptides undergo pH-dependent changes attributed to protonation of -OPO(3)(2-) (dibasic) to -OPO(3)H(-) (monobasic). Bands at approximately 980 and 1080cm(-1) in solution Raman spectra of phosphoserine and phosphothreonine are assigned to the monobasic and dibasic phosphate groups, respectively. Calibrated Raman peak area ratio measurements, performed as a function of pH, are used to determine the corresponding pKa values of 5.6 (phosphoserine) and 5.9 (phosphothreonine). In peptides, the phosphate Raman bands are difficult to distinguish due to interference from other neighboring bands (particularly those derived from aromatic amino acid residues) as well as the relatively low solubility of peptides. Nevertheless, drop coating deposition Raman (DCDR) spectra obtained from 100-microM peptide solutions reveal pH-dependent second derivative features at approximately 980 and 1080cm(-1), which are indicative of phosphate protonation.
磷酸化氨基酸和肽的拉曼光谱会发生pH依赖性变化，这归因于-OPO(3)(2-)(二元)质子化为-OPO(3)H(-)(一元)。磷酸丝氨酸和磷酸苏氨酸溶液拉曼光谱中约980和1080cm(-1)处的谱带分别归属于一元和二元磷酸基团。作为pH函数进行的校准拉曼峰面积比测量用于确定相应的pKa值，磷酸丝氨酸为5.6，磷酸苏氨酸为5.9。在肽中，由于来自其他相邻谱带（特别是那些源自芳香族氨基酸残基的谱带）的干扰以及肽相对较低的溶解度，磷酸拉曼谱带难以区分。然而，从100μM肽溶液获得的滴涂沉积拉曼（DCDR）光谱在约980和1080cm(-1)处显示出pH依赖性二阶导数特征，这表明磷酸发生了质子化。
