Comparative study of protein molecular weights by size-exclusion chromatography and laser-light scattering.
High-performance size-exclusion chromatography (SEC) based on UV-Vis detection is a relative technique for molecular weight determination whereas procedure based on multi-angle laser light scattering (MALLS) is both rapid and absolute. The two methods using recombinant human growth hormone (rHGH) and beta-lactoglobulin samples were compared. A calibration curve for the chromatographic system was generated based on standard proteins and the data were fitted by least squares to a third order polynomial model. The molecular weight from the conventional SEC method for both proteins was higher than the reported values. The molecular weight of rHGH from MALLS was 23.1+/-0.57 and 21.2+/-0.80 kDa using differential refractive index (SEC-MALLS/RI) and UV (SEC-MALLS/UV-Vis) detectors as mass detectors. Both values agree, within experimental error with the molecular weight sequence of rHGH, 22.1 kDa. In contrast, the molecular weight from LS for beta-lactoglobulin was 22.5+/-0.55 kDa by SEC-MALLS/RI and 23.0+/-1.22 kDa by SEC-MALLS/UV-Vis, respectively, values always higher than those supplied by the manufacturer, 18.4 kDa. The reproducibility of the SEC-MALLS/UV-Vis method versus the SEC-MALLS/RI method was performed using the concordance correlation coefficient. The method's reproducibility was accepted by assuming a precision of 98% and a 1% loss in precision.
Oliva
A
A
Departamento Ingeniería Química y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de La Laguna, 38200, Tenerife, Spain
Llabrés
M
M
Fariña
J B
JB
D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013006:Growth Hormone / Q000737:chemistry*; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry*; D007834:Lasers; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012542:Scattering, Radiation
D007782:Lactoglobulins; D011994:Recombinant Proteins; D013006:Growth Hormone