Dharmasiri K, Smith D L
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Anal Chem. 1996 Jul 15;68(14):2340-4. doi: 10.1021/ac9601526.
The rates at which peptide amide hydrogens in folded proteins undergo isotopic exchange are reduced by factors of 10(0)-10(-8) relative to exchange rates at the same peptide linkages in unfolded proteins. To measure the isotopic exchange rates of the most rapidly exchanging peptide amide hydrogens in proteins, a flow-quench deuterium exchange-in step has been added to the protein fragmentation/mass spectrometry method (Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531). Isotopic exchange rates in eight short segments spanning the entire backbone of cytochrome c have been determined for exchange-in times of 0.2-120 s. These results show that the isotopic exchange rates of 10 of the peptide amide hydrogens in cytochrome c are similar to those expected for unfolded cyt c, while the exchange rates for 33 other non-hydrogen-bonded amide hydrogens are much less than expected for unfolded cyt c. Since the isotopic exchange rates of the most rapidly exchanging amide hydrogens in folded proteins are a direct measure of their access to the aqueous solvent, the ability to determine these isotopic exchange rates points to the possibility of using quenched-flow amide hydrogen exchange and mass spectrometry as a tool for identifying protein surfaces involved with binding.
相对于未折叠蛋白质中相同肽键处的交换速率，折叠蛋白质中肽酰胺氢进行同位素交换的速率降低了10(0)-10(-8)倍。为了测量蛋白质中交换最快的肽酰胺氢的同位素交换速率，在蛋白质片段化/质谱法中增加了流动淬灭氘交换步骤（Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531）。已确定了跨越细胞色素c整个主链的八个短片段在0.2-120秒的交换时间内的同位素交换速率。这些结果表明，细胞色素c中10个肽酰胺氢的同位素交换速率与未折叠的细胞色素c预期的速率相似，而其他33个非氢键结合的酰胺氢的交换速率远低于未折叠的细胞色素c预期的速率。由于折叠蛋白质中交换最快的酰胺氢的同位素交换速率是其与水溶剂接触程度的直接衡量指标，能够确定这些同位素交换速率表明有可能将淬灭流动酰胺氢交换和质谱法用作识别参与结合的蛋白质表面的工具。
