Stachurska Karolina, Antosiewicz Jan M
Biophysics Division, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5 St., 02-093 Warsaw, Poland.
ACS Omega. 2024 Oct 16;9(43):43998-44004. doi: 10.1021/acsomega.4c08874. eCollection 2024 Oct 29.
The structural changes induced by the addition of sodium dodecyl sulfate (SDS) in chymotrypsin and chymotrypsinogen were studied by the stopped-flow kinetic method with tryptophan fluorescence observation of the transients. Four fluorescence excitation wavelengths were used: 222, 260, 280, and 295 nm. It was found that the recorded transients were dependent on the excitation wavelength. The difference emission spectra between the complex and the free enzyme recorded for different excitation wavelengths are different. They contain a positive limb of fluorescence enhancement around 380 nm and a limb of fluorescence quenching around 340 nm. Their relative sizes depend on the excitation length, which fully explains the kinetic observations and proves that the contribution of tryptophans distributed at different sites within the protein molecule to its total fluorescence depends on the excitation wavelength. This is an important and novel finding that goes beyond the well-known fact that tryptophans distributed at different sites in the protein molecule have different fluorescence intensities.
采用停流动力学方法并通过观察色氨酸荧光瞬态，研究了在胰凝乳蛋白酶和胰凝乳蛋白酶原中添加十二烷基硫酸钠（SDS）所引起的结构变化。使用了四个荧光激发波长：222、260、280和295纳米。发现所记录的瞬态依赖于激发波长。针对不同激发波长记录的复合物与游离酶之间的差异发射光谱各不相同。它们在380纳米左右包含荧光增强的正向峰以及在340纳米左右的荧光猝灭峰。它们的相对大小取决于激发波长，这充分解释了动力学观测结果，并证明了分布在蛋白质分子内不同位点的色氨酸对其总荧光的贡献取决于激发波长。这是一项重要且新颖的发现，超越了蛋白质分子中分布在不同位点的色氨酸具有不同荧光强度这一众所周知的事实。
