Ramsay G, Ionescu R, Eftink M R
Department of Chemistry, University of Mississippi, University 38677, USA.
Biophys J. 1995 Aug;69(2):701-7. doi: 10.1016/S0006-3495(95)79945-3.
In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.
在之前的一篇论文中（拉姆齐和埃夫廷克，《生物物理杂志》66:516 - 523），我们报道了一种改进型分光光度计的研发，该仪器能够几乎同时进行圆二色性（CD）和荧光测量。这种配置允许在单个实验中收集多个数据集，从而节省时间和材料，并改善不同类型测量之间的相关性。通过对几种不同蛋白质系统进行热变性实验，展示了该仪器的实用性。这种CD/荧光分光光度计已通过与注射泵和pH计连接进行了进一步改进。这种配置允许在监测样品的CD、吸光度、荧光和光散射特性变化的同时，进行配体、pH和化学变性滴定实验。我们的数据采集程序还能够在记录数据之前检查信号是否已接近平衡。为了进行pH滴定，我们开发了一种程序，该程序在反馈回路中使用来自pH计的信号，以便在均匀间隔的pH值下收集数据。我们通过研究酸pH值和添加盐酸胍诱导的抹香鲸脱辅基肌红蛋白的展开，展示了该仪器的用途。
