Accurate determination of protein size and molecular weight is important for obtaining information on differences in molecular shape, such as between globular and fibrous proteins, as well as on the subunit composition of large proteins. In addition, molecular weight information is useful for characterizing protein–protein associations, such as immune complexes and complexes formed between enzymes and their inhibitor proteins. Several methods for determining the molecular weight of biopolymers, including proteins, are known, such as osmometry, light scattering, and sedimentation equilibrium. However, these techniques often require complicated and time-consuming procedures. In this application, protein was analyzed by PROTEIN KW-803, a silica-based aqueous SEC (GFC) column combined with multi-angle light scattering detection (SEC/MALS). Figure 1 plots the relationship between molecular weight measured by MALS and elution volume, while the chromatograms show the signals obtained with a refractive index (RI) detector. Figure 2 shows a calibration curve constructed from the molecular weight and elution volume of each protein determined by MALS, together with the actual elution positions of the proteins. For bovine serum albumin (BSA), a neutral protein, the molecular weight obtained by MALS in Figure 1 is in good agreement with the known value, and therefore the BSA data point lies on the calibration curve. In contrast, lysozyme, a basic protein, elutes later than expected, while α₁-acid glycoprotein, an acidic protein, elutes earlier than expected. As a result, both proteins deviate from the calibration curve. Molecular weights estimated from a calibration curve appear smaller than the true values for proteins that elute later, and larger than the true values for proteins that elute earlier. Consequently, if an unknown protein shows elution behavior that deviates from the calibration curve, the estimated molecular weight may differ from its actual value. For these reasons, combining SEC with MALS, which enables direct determination of molecular weight, is effective for obtaining accurate molecular weight information for macromolecules. As an example demonstrating the usefulness of MALS, a small peak appearing before the main peak is observed in the BSA chromatogram in Figure 1. The molecular weight determined by MALS for this peak is approximately twice that of BSA, suggesting that the peak corresponds to a BSA dimer.

Sample:
BSA (Sigma)
α₁ Acid glycoprotein, Orosomucoid (Sigma)
Lysozyme (Seikagaku Kogyo Co., Ltd.)

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