Interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry for analysis of proteins.
Molecular weights (MWs) of different proteins were determined by interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry (IEC-LALLS). A high-performance strong cation-exchange column was used to elute proteins using fast (5 min) and conventional (15-30 min) gradients. The eluted proteins were characterized on-line by determining their MWs using LALLS. The specific refractive index (RI) increment (dn/dc) and the RI of the solvent used over the gradient range were determined off-line and used to calculate the absolute weight-average MWs. Four proteins, ribonuclease A, alpha-chymotrypsinogen A, trypsinogen and beta-lactoglobulin A (beta-LACT) were studied. Accurate MWs were obtained for all the proteins using fast and conventional gradients, except for beta-LACT, which aggregated as a function of the gradient employed. The degree of aggregation of beta-LACT increased as the rapidity of the gradient was increased over a fixed gradient range. This study indicated that it is possible to separate and characterize proteins rapidly using IEC-LALLS.
Mhatre
R M
RM
Department of Chemistry, Northeastern University, Boston, MA 02115.
Krull
I S
IS
D002852:Chromatography, Ion Exchange / Q000379:methods*; D007834:Lasers; D008027:Light; D011506:Proteins / Q000032:analysis*; D012031:Refractometry; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis
D011506:Proteins