使用SEC-MALS技术来表征血红蛋白

Hemoglobin is the key protein in the red blood cells of mammals, playing the essential role in transporting oxygen from the lungs to respiring tissue in the body. Subsequent to the release of oxygen, carbon dioxide binds to hemoglobin to be released out of the body.

Instrumentation

Human hemoglobin is composed of four polypeptides, wherein the tetramer equilibrates with a dimer form based on the conditions. A combination of size-exclusion chromatography (SEC), multi-angle light scattering (MALS), Optilab DSP interferometric refractometer, and a miniDAWN detector is used for hemoglobin characterization, enabling to readily acquire data on absolute molar masses and molar mass distributions.

分析和结果

The hemoglobin molar mass is plotted against the elution time as shown in Figure 1. The molar mass across the peak is not consistent, which is probably owing to an equilibrium shift from a tetramer to a dimer that occurred during the chromatography run.

It may be difficult to differentiate regular peak extending from the small polydispersity of the sample by means of conventional column calibration. However, it is easier with the SEC-MALS measurement.

Figure 1.使用两列从SEC获得的血红蛋白的摩尔质量与洗脱时间，并与MALS检测结合。

牛血清白蛋白（BSA）是一种标准蛋白，通常用于柱校准。图2显示了血红蛋白与BSA的摩尔质量与洗脱时间图的覆盖。尽管两种蛋白质的摩尔质量几乎在同一范围内，但它们的形状和相应的洗脱时间完全不同。然而，BSA在血红蛋白之前恢复了，因为与血红蛋白相比，它的紧凑结构较低。

Figure 2.BSA and hemoglobin (proteins with similar molar masses) elute at different volumes, due to the more compact structure of hemoglobin.

Conclusion

If BSA is used as a calibration standard as in many laboratories, there is a possibility for the hemoglobin molar mass to be significantly underestimated. However, it is possible to avoid all of the assumptions and erroneous conclusions by coupling the chromatography with a miniDAWN or DAWN.

This information has been sourced, reviewed and adapted from materials provided by Wyatt Technology.

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