Miclo L, Perrin E, Driou A, Mellet M, Linden G
Applied Biochemistry Laboratory (associated with INRA), Science Faculty, Henri Poincaré University of Nancy I, Vandoeuvre-lés-Nancy, France.
Int J Pept Protein Res. 1995 Aug;46(2):186-92. doi: 10.1111/j.1399-3011.1995.tb01335.x.
A method for the simultaneous determination of the ratios of the three aromatic amino-acid residues in peptides was set up in acidic conditions. Binary and ternary mixtures of these amino acids were prepared, and first- and second-derivative spectra then calculated from their 0.1 nm resolution spectra between 240 and 320 nm. Certain spectral bands were chosen to differentiate tyrosine from tryptophan on the first-derivative spectra, and phenylalanine from tyrosine and tryptophan on the second-derivative spectra. Variation of the amplitude of the chosen bands was shown to be a linear function of the ratio of the aromatic amino acids in the mixture. This technique was validated with peptides whose sequence was known. The difference between theoretical and experimentally determined ratios was lower than 10%. Since the results are obtained as ratios, neither the concentration nor the nature of the peptide has to be known. The feasibility of application using a photodiode array detector with high resolution in reversed-phase high-performance liquid chromatography is discussed.
建立了一种在酸性条件下同时测定肽中三种芳香族氨基酸残基比例的方法。制备了这些氨基酸的二元和三元混合物，然后根据其在240至320 nm之间分辨率为0.1 nm的光谱计算一阶和二阶导数光谱。在一阶导数光谱上选择某些光谱带来区分酪氨酸和色氨酸，在二阶导数光谱上区分苯丙氨酸与酪氨酸和色氨酸。所选谱带幅度的变化被证明是混合物中芳香族氨基酸比例的线性函数。该技术用序列已知的肽进行了验证。理论值与实验测定值之间的差异低于10%。由于结果是以比例形式获得的，因此无需知道肽的浓度和性质。讨论了在反相高效液相色谱中使用具有高分辨率的光电二极管阵列检测器进行应用的可行性。
