Characterization of Oligomer Formation of Surfactant Protein-D (SP-D) Using AF4-MALLS

Background: Surfactant protein-S (SP-D) is a naturally occurring lung protein with the potential to treat pulmonary infections. A recombinant surfactant protein-D (SP-D) has been produced and was previously found to exist in multiple oligomeric states. Introduction: Separation and characterization of interconverting oligomeric states of a protein can be difficult using chromatographic methods, so an alternative separation technique was employed for SPD to characterize the different association states that exist. Methods: Samples of SP-D were analyzed using asymmetrical flow field-flow fractionation (AF4) using UV and multi-angle laser light scattering (MALLS) detection. The AF4 method appears to be able to separate species as small as the monomer up to the dodecamer (the dominant species) to much larger species with a molar mass greater than 5 MDa. Results: Consistent elution of four distinct peaks was observed after repeated injections. The largest species observed under the last peak (labeled as Peak 4) were termed “unstructured multimers” and were resolved fairly well from the other species. The AF4-MALLS data suggest that only a small fraction of Peak 4 truly corresponds to high molar mass unstructured multimers. All other peaks demonstrated significant molar mass homogeneity consistent with AFM results. Conclusion: AF4-MALLS technology appears to be a powerful analytical approach to characterize the complex and dynamic interplay among different protein oligomeric species of SP-D in an aqueous solution.