Michael Kunitani

Reversed-phase chromatography of interleukin-2 muteins.

Human recombinant interleukin-2 (IL-2) and related species have been characterized by chemical modifications, tryptic digestion, and cyanogen bromide digestion. The oxidation states of the cysteines and methionines in several IL-2 muteins have been determined. Reversed-phase high-performance liquid chromatography allowed us to distinguish the modifications in these muteins and to correlate retention behavior with their structure.

On-line characterization of polyethylene glycol-modified proteins

Abstract An high-performance liquid chromatographic method has been developed which simultaneously determines three critical physical properties of polyethylene glycol (PEG)-modified proteins: molecular size, polymer distribution and weight composition. With both UV and refractive index (RI) detectors in series, size-exclusion chromatography (SEC) is used to separate the PEG-protein species according to size. The size analysis of these PEG-proteins is predicted to be accurately calibrated with the viscosity radius (universal calibration), which compensates for the shape differences between PEG and protein structures. The heterogeneity of the PEG-protein grafted copolymer is represented by the polymeric term “polydispersity”, which describes the size distribution. Separate SEC calibrations of the PEG and the protein used for conjugation allow a determination of the weight composition of the PEG-protein (weight PEG/weight protein) by combining UV and RI chromatograms of a PEG-protein sample. This compositional analysis is validated through independent and direct measurement of the PEG on a PEG-protein via acid hydrolysis and quantitative SEC. Comparisons of compositional analysis of PEG-protein with sodium dodecyl sulfate polyacrylamide gel electrophoresis densitometry demonstrate that gel analysis of some proteins is misleading.

Practical on-line determination of biopolymer molecular weights by high-performance liquid chromatography with classical light-scattering detection

Abstract Most biopolymer molecules are much smaller than the wavelength of light used in classical light-scattering experiments ( ca. 500 nm), and thus the simple Rayleigh equation and a 90° light-scattering photometer are sufficient to determine their molecular weight. In combination with high-performance liquid chromatography (HPLC), it is demonstrated that a simple HPLC fluorimeter can be used as a 90° light-scattering detector for biopolymer molecular weight determinations. To simplify data handling, only relative molecular weights are measured. Three mathematical assumptions are adopted, and their validity for proteins is shown. To place the work in perspective, the relative advantages and limitations of this 90° light-scattering detector are compared with the more commonly used low-angle laser light-scattering detector. Two examples of protein molecular weight determinations are given to illustrate the broad utility of 90° classical light scattering to the study of biopolymer structures and interactions.

Reversible subunit dissociation of tumor necrosis factor during hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) of recombinant tumor necrosis factor (TNF) results in reversible dissociation of the quaternary protein structure yielding separation of trimer and monomer peaks. Gel electrophoresis, size exclusion, fluorescence polarization and rechromatography were used to identify the trimeric and monomeric species. Relative amounts of these peaks varied as a function of temperature and column contact time. When TNF was chromatographed in the presence of partially proteolyzed [14 kilodalton (kD)] TNF, two additional peaks, identified as the 14-kD monomer and heterotrimer of TNF and the 14-kD fragment, appeared. Rechromatography of this heterotrimer produced TNF monomer and 14-kD peaks establishing that the multiple peak pattern in HIC was due to quaternary dissociation. Incubating TNF in denaturants prior to non-denaturing size-exclusion chromatography resulted in apparent protein unfolding. However, free, undenatured monomer was not observed. We conclude that TNF is most likely a trimer, which is tightly held together by hydrophobic forces, and that the tertiary structure of the monomer is stabilized through this subunit association. The hydrophobicity of the sorbent surface mediates reversible dissociation of the trimers to monomers through hydrophobic stabilization of the monomeric tertiary structure. After elution, the TNF monomers reassociate to form the native TNF trimer.