Temperature-Insensitive Near-Infrared Method for Determination of Protein Concentration during Protein Crystal Growth

• Published in: Analytical chemistry (Washington) 2000-10, Vol.72 (20), p.4985-4990
• Main Authors: Olesberg, J. T, Arnold, M. A, Hu, Shih-Yao B, Wiencek, John M
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Proteins; Proteins - chemistry; Solvents; Spectroscopy, Near-Infrared - methods; Spectrum analysis; Temperature
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac000406u

A temperature-insensitive method for measuring protein concentration in aqueous solutions using near-infrared spectroscopy is described. The method, which is based on identification of the net analyte signal of single-beam spectra, can be calibrated using a single protein absorbance measurement and is thus well suited for crystallization monitoring where the quantity of protein is limited. The method is applied to measurements of glucose-isomerase concentration in a sodium phosphate buffer that is actively varied over the temperature range of 4−24 °C. The standard error of prediction using the optimized spectral range of 4670−4595 cm-1 is 0.12 mg/mL with no systematic trend in the residuals with solution temperature. The method is also applied to previously collected spectra of hen egg-white lysozyme and yields a standard error of prediction of 0.14 mg/mL. Spectra sampled at discrete wavelengths can also be used for calibration and prediction with performance comparable to that obtained with spectral bands. A set of four wavelengths are identified that can be used to predict concentrations of both proteins with a standard error less than 0.14 mg/mL.