Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules.

Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications.

The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.

Glutaraldehyde modification of recombinant human hemoglobin alters its hemodynamic properties.

Many cell-free hemoglobin solutions designed as oxygen-carrying therapeutics produce a hypertensive effect in animals. The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by glutaraldehyde would attenuate the hypertensive response. Two products of the cross-linking reaction were isolated, a glutaraldehyde-derivatized monomer (mono-glxrHb) and a glutaraldehyde cross-linked polymer (poly-glxrHb), and evaluated for their effects on systemic hemodynamics in conscious rats. Administration of rHb1.1 caused a mean arterial pressure elevation of approximately 20 mm Hg and an increase in total peripheral resistance of approximately 30%. Administration of mono-glxrHb induced changes in mean arterial pressure and vascular resistance that were significantly diminished relative to those observed with rHb1.1. Poly-glxrHb elicited a mean arterial pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same as the response to mono-glxrHb. These results suggest that rHb peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and that the effect of glutaraldehyde polymerization is likely due to surface modification and/or intramolecular cross-linking, rather than an increase in molecular size.

Effects of Tween 80 and sucrose on acute short-term stability and long-term storage at -20 degrees C of a recombinant hemoglobin.

The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to recombinant hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze-thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze-thaw induced aggregation. In contrast to the acute stability studies, long-term storage at -20 degrees C induced aggregation and methemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested methemoglobin formation during the long-term -20 degrees C storage. Specific binding of Tween 80 to the hemoglobin was not observed using 16-doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the hemoglobin from reaching the air-liquid interface or the liquid-surface interfaces. The reduction in methemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.

Photolabeling evidence for calcium-induced conformational changes at the ATP binding site of scallop myosin.

A change in the conformation of the active site of scallop myosin under the influence of regulatory amounts of Ca2+ has been identified by use of the ADP photoaffinity analog 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP). NANDP, trapped at the active site with Mn2+ and vanadate, photolabeled preferentially Arg-128 of the heavy chain in the absence of added Mg2+ and Ca2+ [Kerwin, B. & Yount, R. (1992) Bioconjugate Chem. 3, 328-336]. However, addition of 2 mM Mg2+ and regulatory amounts of Ca2+ (0.01-1 microM) shifted the predominant labeling to Cys-198 of the heavy chain in a Ca(2+)-dependent manner. This Ca(2+)-dependent change in the photolabeling pattern was absent when the regulatory light chains were removed or when the unregulated head (subfragment 1) was examined under similar conditions. These results demonstrate that both Arg-128 and Cys-198 are part of the purine binding site which undergoes a conformational change in response to Ca2+ binding to the regulatory domain.

Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin.

The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP) was used to photolabel the ATP binding site of scallop myosin. Approximately 1 mol of NANDP per mol of myosin was trapped at the active site by complexation with vanadate and manganese. ADP, but not AMP, inhibited trapping of NANDP. The trapped NANDP photolabeled up to 37% of the myosin upon UV irradiation. Papain subfragment-1 prepared from the photolabeled myosin was digested with trypsin, and the major photolabeled tryptic peptides were isolated by reversed-phase HPLC. The amino acid sequence of the major labeled peptide was X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys, where X represents the photolabeled amino acid Arg128. Previously, Trp130 of rabbit skeletal muscle myosin has been shown to be photolabeled by NANDP [Okamoto, Y., and Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1580]. Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with Arg128 in an equivalent position as Trp130. These results suggest that the composition of the purine binding site is analogous in both myosins and that Arg and Trp play a similar role in binding ATP, despite the marked differences of their side chains.

Photochemical mapping of the active site of myosin.

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the gamma-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.