Hemoglobin–albumin cross-linking with disuccinimidyl suberate (DSS) and/or glutaraldehyde for blood substitutes

Figures & data

Figure 1. Chemical structure of the polymerization reagent used DSS.

Figure 2. SDS–PAGE of polyHb. 1: native Hb; 2: 1Hb 8GL; 3: 1Hb 2BSA 8GL; 4: 1Hb 2BSA 5DSS; 6: 1Hb 2BSA 5DSS 8GL; and M: marker.

Figure 3. Size exclusion chromatograms for selected samples.

Figure 4. UV–vis spectra of GL-polymerized Hbs as prepared and after incubation at 37°C for 4 h.

Table I. Autooxidation rates (values compared with native Hb), rate constants (K_d), and Hill coefficients (n) for polyHb and native Hb.

Protein	Autooxidation	Oxygen affinity (P50, mmHg)	Hill coefficient
Native Hb	–	23.8	1.9
1Hb 8GL	> 1.8x	2.6 (< 9.1x)	1
1Hb 2BSA 8GL	< 2.1x	19.0 (< 1.25x)	0.7
1Hb 2BSA 5DSS	> 1.4x	12.7 (< 1.87x)	0.7
1Hb 2BSA 5DSS 2GL	> 1.5x	18.7 (< 1.27x)	0.9

Figure 5. The reaction of GL-polymerized Hb (A) and DSS-polymerized Hb (B) with H₂O₂. Conditions: room temperature, PBS 10 μM protein, 80 μM H₂O₂.

Figure 6. EPR spectra of 200 μM protein treated with 500 μM H₂O₂ in PBS and frozen at 30 s after mixing.