蛋白质的圆二色光谱测量中圆二色值不确定度评估——以细胞色素C为例

Spectral Magnitude Uncertainty in Measurement of Protein Circular Dichroism Spectra-An Empirical Study on Cytochrome C

圆二色(CD)光谱是一种公认的用于测量蛋白质及其二级结构的生物物理技术,不但被广泛用于测量蛋白质的二级结构,而且可以检测二级及更高级结构的变化,同时用于科学研究和蛋白质产品如生物制药的质量控制。由于对CD光谱测量中的误差来源定量理解有限,对光谱进行客观比较仍具有挑战性。统计方法可用于比较,但不提供处理系统性以及随机性误差的机制。在任意两台仪器中进行的CD测量,即使在可比较的条件下,相同样品的光谱幅度(elipticity椭圆度,CD scale,也称圆二色值)或波长也可能存在细微差异。光源、最终的光度输出和各个仪器之间入射光偏振的微小差异是导致圆二色值或波长产生差异的可能原因。分析蛋白质CD光谱取决于原始CD数据的质量,CD解卷积分析强烈依赖于CD光谱中谱峰的圆二色值。因此,以一种α-螺旋为主导的蛋白质—细胞色素C为实验对象,在对CD光谱仪进行校准的前提下,对其浓度为0.05 mg·mL^(-1)的水溶液进行圆二色光谱测定,继而建立了0.05 mg·mL^(-1)细胞色素C水溶液的CD光谱测量模型,评估了其在波长222 nm谱峰圆二色值的不确定度。圆二色值的不确定度来源主要有测量重复性、校准溶液及蛋白溶液浓度的不确定度、比色皿光程长的不确定度等。经过仪器校准后,综合考虑这些不确定来源,得到0.05 mg·mL^(-1)细胞色素C水溶液在波长为222 nm处谱峰的圆二色值及不确定度为(-4.53±0.54)mdeg,k=2。通过不确定度评估,发现在不确定分量中较为显着的为1 mm比色皿光程长不确定度和溶液配置过程引入的不确定度分量,设法消除或降低这些因素的影响,可以改进测量方法分析测量过程,以实现CD光谱的客观比较,提高CD谱图的可比性和可靠性,为开展圆二色光谱测量的实验室间比对提供实验参考。

Circular dichroism(CD)spectroscopy is a well-established biophysical technique used to measure protein and its secondary structure and to detect changes in secondary and higher orders of structure for applications in research and the quality control of protein products such as biopharmaceuticals.However,objective comparison of spectra is challenging because of a limited quantitative understanding of the sources of error in the measurement.Statistical methods can be used for comparisons but do not provide a mechanism for dealing with systematic and random,errors.CD measurements in any two instruments may often present slight differences in spectral magnitude or wavelength,even for the same sample under comparable conditions.The small disparities between the polarization of the incident light from each instrument,light source,and final lamp output are examples of the variables that can produce such differences.On the other hand,the structural information acquired with the CD method can sometimes be hampered by the poor quality of the original CD data,and CD deconvolution analysis strongly depends on the spectral intensity.Here a helix predominate protein—cytochrome C was taken as the experimental object,and CD spectroscopy was used to measure the concentration of 0.05 mg·mL^(-1)cytochrome C aqueous solution after instruments were typically calibrated using standards.And then,a measurement model for CD spectroscopy of 0.05 mg·mL^(-1)Cytochrome C aqueous solution was presented,incorporating the principal sources of uncertainty to derive an uncertainty budget of spectral magnitude in wavelength 222 nm.The uncertainties of spectral magnitude were from measurement repeatability,concentration uncertainty of calibration solution and protein solution,the uncertainty of cell length of the cuvette,etc.After calibrating the instrument,these sources of uncertainty were comprehensively considered,and the magnitude uncertainty of 0.05 mg·mL^(-1)cytochrome C aqueous solution at the wavelength of 222 nm was(-4.53±0.54)mdeg,k=2.The uncertainty,evaluation found that the uncertainty of 1 mm cuvette cell length and the solution preparation process account for a significant part of the uncertainty component.Eliminating or reducing the impact of these factors can improve the measurement method to analyze the measurement process to achieve an objective comparison of CD spectra and improve the comparability and reliability of CD spectra.This work also provides an experimental reference for the interlaboratory comparison of circular dichroism measurement.