Polymerization of modified diaspirin cross-linked hemoglobin (DCLHb) with 1,6-bismaleimic-hexane

Figures & data

Figure 1. Conversion of amino group of DCLHb to thiol group with 2-IT.

Figure 2. Structure of 1,6-BMH.

Figure 3. SDS-PAGE. (A–D) represent the elution of MW standards, purified Hb, DCLHb, and DCLHb-SH, respectively. E and F are polyDCLHb (cross-linked by 1,6-BMH).

Figure 4. UV-Vis wavelength scanning of Hb, DCLHb-SH, and polyDCLHb.

Figure 5. SEC-MALLS of polyDCLHb (ratio of 2-IT to Hb).

Table 1. MW distribution of polyDCLHb by SEC-MALLS.

Entry	Retain time (min)	MW (g/mol)
Peak 1	15.744–18.004	2.649 × 10^5
Peak 2	18.051–19.913	1.307 × 10^5
Peak 3	20.007–21.483	6.894 × 10^4
Peak 4	21.530–22.536	3.763 × 10^4

Figure 6. HPLC of the products. (a) purified Hb, (b) DCLHb, and (c) polyDCLHb mobile phase 1 mol/L MgCl₂.

Table 2. The constituents and corresponding proportion of the chemical modified Hb.

Entry	Retain time (min)	Peak area	Peak area (%)
HMWs	17.484	154,341	7.14
PolyDCLHbs	18.660	416,739	19.29
Tetrimers	20.726	13,203,13	61.11
Dimers	21.704	269,332	12.46

Table 3. The proportion of polyDCLHb in the polymerization of DCLHb with 1,6-BMH with varial quantity of bromoacetic acid and 2-IT.

Entry	Molar ratio of bramoacetic acid to Hb	Molar ratio of 2-IT to Hb	Retain time (min)	Peak area	Peak area (%)
A1	3	1.5	18.708	268,012	14.33
A2	3	2	18.659	298,208	15.15
A3	3	3	18.704	289,441	14.95
B1	2	1.5	18.715	290,138	17.09
B2	2	2	18.713	385,831	18.16
B3	2	3	18.708	374,684	17.92

Figure 7. Oxygen binding curve of native hemoglobin was measured using a Hemox analyzer at 37 °C in PBS, pH 7.4. Vertical axis is the fraction of hemoglobin sites to which oxygen is bound. The partial oxygen pressure at 50% saturation is expressed in mmHg.

Figure 8. Oxygen binding curve of polyDCLHb was measured using a Hemox analyzer at 37 °C in PBS, pH 7.4 (2-IT 2 mmol/L, cross-linking agent 1 mmol/L).