Temperature-insensitive near-infrared method for determination of protein concentration during protein crystal growth.
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 degrees 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.
Olesberg
J T
JT
Department of Chemistry and the Optical Science and Technology Center, The University of Iowa, Iowa City 52242, USA.
Arnold
M A
MA
Hu
S Y
SY
Wiencek
J M
JM
D011506:Proteins / Q000737:chemistry*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D013696:Temperature
D011506:Proteins