Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory.
Size exclusion chromatography (SEC) coupled with "on-line" laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection provides an elegant approach to determining the molecular weights of proteins and their complexes in solution. SEC serves solely as a fractionation step to minimize the ambiguity that otherwise can result from the fact that light scattering provides the weight-average molecular weight (MW) of all species in solution. Our goal is to establish realistic expectations for MW determination using LS coupled with SEC, define sample requirements, and identify possible limitations of SEC/LS analysis. Analyses of 14 protein standards that range from 12 to 475 kd suggest that the molecular weights of native proteins may be determined in a single SEC/LS experiment with an accuracy of +/-5%. The MW determination depends only on the downstream LS and RI detectors, and it is independent of elution position. Unusual elution because of nonglobular shape or interaction with the SEC support has no impact on the MW determination by SEC/LS. With the instrument configuration that was used, the optimal amount of protein needed for SEC/LS is about 50 g for proteins with molecular weight greater than 40 kd. However, analyses of ovalbumin and transferrin demonstrate that even 10 g is sufficient to determine the MW with an error of less than +/-6%. Although SEC/LS has some limitations, such as proteins that contain chromophores whose absorption spectrum overlaps that of the emission spectrum of the laser, it represents a fast and robust approach to determining MW and to monitoring protein oligomerization in solution.
Folta-Stogniew
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HHMI Biopolymer Laboratory and W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT, USA. Ewa.Folta-Stogniew@yale.edu
Williams
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KR