Mhatre R M, Krull I S
Department of Chemistry, Northeastern University, Boston, MA 02115.
J Chromatogr. 1992 Feb 7;591(1-2):139-48. doi: 10.1016/0021-9673(92)80231-i.
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.
通过将梯度洗脱离子交换色谱法与低角度激光光散射光度法（IEC-LALLS）联用测定不同蛋白质的分子量（MWs）。使用高性能强阳离子交换柱，采用快速（5分钟）和常规（15 - 30分钟）梯度洗脱蛋白质。通过使用LALLS测定洗脱蛋白质的MWs对其进行在线表征。特定折射率（RI）增量（dn/dc）和梯度范围内使用的溶剂的RI离线测定，并用于计算绝对重均分子量。研究了四种蛋白质，核糖核酸酶A、α-胰凝乳蛋白酶原A、胰蛋白酶原和β-乳球蛋白A（β-LACT）。除了β-LACT，使用快速和常规梯度都能获得所有蛋白质的准确MWs，β-LACT会随着所用梯度而聚集。在固定梯度范围内，随着梯度速度的增加，β-LACT的聚集程度增加。该研究表明，使用IEC-LALLS可以快速分离和表征蛋白质。
