Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic

Abstract

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.

Figures

FIGURE 1

Schematic representation of the reaction to produce P5K6. In addition to the specific reaction of the two β-subunit Cys-93 residues to produce P5K2, an additional four to five lysine surface residues are modified by thiolation, followed by the maleimide-PEGylation reaction.

FIGURE 2

Scattered intensities of Hb at 5 g/l (pink) and 31 g/l (blue) and of P5K6 at 5 g/l (green) and 21 g/l (red) (A). Structure factors of Hb (blue), P5K2 (green), and P5K6 (red) (B).

FIGURE 3

Processed experimental scattering data (from top to bottom: Hb, P5K2, and P5K6), and the scattering calculated from the models. Solid lines and open circles are the fits by ab initio models from GASBOR and MONSA, respectively; dashed lines are scattering patterns computed from the crystallographic models of oxyHb or deoxyHb. The curves are displaced along the abscissa axis for clarity (A). Distance distribution functions of Hb (blue), P5K2 (green), and P5K6 (red); the arrow marks the maximum appearing in the PEG-containing constructs at r = 75 Å (B).

FIGURE 4

Representative GASBOR models of Hb (A), P5K2 (C), and P5K6 (D). Dark blue beads show the protein and/or PEG dummy residues, light gray beads represent the dummy waters imitating the solvation shell. Structural alignment of the Hb model (shown as a transparent surface) to the crystal structure of oxyHb (shown as a ribbon model) (B).

FIGURE 5

Low-resolution multiphase model of PEGylated Hb constructed ab initio using the program MONSA. Cyan beads belong to Hb, red beads belong to the two PEG chains in P5K2, and yellow beads are the rest of the four to five PEG chains in P5K6; bead radius, 2.5 Å (A and C). In the right panel (B and D), the Cα chains in oxyHb (PDB entry 2DN1) were positioned by Supcomb (24) to best match the beads depicting the Hb volume (the latter are not displayed for clarity). The Cys-β93 residues are shown in the crystal structure in purple. Also in panels B and D, the red beads were made larger to better see them; in panels A and C, they are of equal size and thus masked by yellow beads. The bottom view is rotated counterclockwise by 90° around the vertical axis.

FIGURE 6

Hydrophobicity plot for Hb (34). The hydrophobicity score is shown for residue numbers aligned with subunit helical segments.