Copolymerization of recombinant Phascolopsisgouldii hemerythrin with human serum albumin for use in blood substitutes

Figures & data

Figure 1. The three-dimensional structure of the P. gouldii Hr (PDB ID: 1I4Y) and HSA (PDB ID: 1GNJ); lysine residues are colored in green; the two layers of four subunits of Hr octamer are colored in red and pink, respectively; di-iron centers are represented as blue spheres.

Figure 2. 15% SDS-PAGE gel of the reaction mixtures of copolyHr-HSA (lanes a) and of polyHr + polyHSA (lanes b) at 0.15 mM Hr, 8 mM GL, and varying HSA concentrations (listed in Table 1).

Table 1 . Protein and lysine residue concentrations in the GL-polymerization reaction mixtures subjected to the 15% SDS-PAGE as shown in Figure 1. Hr concentration was kept constant at 150 μM (monomer basis) in all reaction mixtures.

[Hr]Lys:[HSA]Lys	5	2	1	0.5	0.25
HSA concentration (μM)	5.6	14.0	28.0	55.9	111.9

Figure 3. Gel filtration size exclusion chromatograms of 6 mM GL (co)polymerized samples.

Figure 4. 15% SDS-PAGE of GL-copolymerized Hr-HSA (lanes a) and mixtures of separately GL-polymerized Hr and HSA (lanes b); reaction conditions: 0.15 mM Hr, [Hr]_Lys: [HSA]_Lys = 2:1 (∼14 μM HSA), GL concentrations varying from 2 to 10 mM as indicated by the number in the labels above the lanes.

Figure 5. Gel filtration chromatograms of Hr-HSA copolymers; reaction conditions: 0.15 mM Hr, [Hr]_Lys:[HSA]_Lys = 2:1 (∼14 μM HSA), GL in concentrations varying from 2 to 10 mM.

Figure 6. UV–vis absorption spectra of Hr-HSA (co)polymerization mixtures; reaction conditions: 0.15 mM Hr, [Hr]_Lys:[HSA]_Lys = 2:1 (∼14 μM HSA), GL in concentrations varying from 2 to 10 mM (note: neither HSA nor polyHSA absorb in the visible region of the spectrum).

Figure 7. Anion-exchange (left panel) and gel filtration size exclusion (right panel) chromatograms of GL-polymerized Hr and GL-co-polymerized Hr-HSA; reaction conditions: 0.15 mM Hr, [Hr]_Lys: [HSA]_Lys = 2:1 (∼14 μM HSA), GL concentrations varying from 2 to 6 mM. A, B, and C labels in the left panel correspond to fractions A, B, and C in the right panel.

Figure 8. UV–vis absorption spectra of the oxy forms of Hr, GL-polymerized Hr, and co-polymerized Hr-HSA.

Table 2. Estimated values of dioxygen affinity (P₅₀) and half-life of auto-oxidation (t_1/2) of unmodified and GL-derivatized Hrs and Hr-HSA GL-copolymers; reaction conditions: 0.15 mM Hr, [Hr]_Lys:[HSA]_Lys = 2:1 (∼14 μM HSA), GL in concentrations as indicated (2 or 4 mM).

	P50 (mmHg)	t1/2 (min)
Hr	9	670
Hr + HSA	11	670
polyHr/2	17	370
CopolyHr-HSA/2	24	390
CopolyHr-HSA/4	25	280
Red blood cell Hb (Eike and Palmer 2004)	28	–

Figure 9. Dioxygen saturation curves for GL-polymerized Hr and copolymerized GL-Hr-HSA.

Figure 10. TEM images of the nHr, polyHr, and copolyHr in MOPS (7.4) solution.

Eike JH, Palmer AF. 2004. Effect of Cl- and H + on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes. Biotechnol Prog. 20:1543–1549.

 PubMed Web of Science ®Google Scholar