Preparation of Snopeghb

To utilize SNO-Hb as an oxygen carrier, we have developed a pyridoxalated and pegylated SNO-Hb derivative having low oxygen affinity and an optimum plasma residence time. The preparation steps are schematically illustrated in Figure 1. The detailed preparation method is described elsewhere5. Briefly, human Hb purified from outdated hman red cell products was mixed with pyridoxal-5'phosphate and pyridoxalation was started by the addition of sodium borohydrate under anaerobic conditions. For the pegylation of pyridoxalated Hb, the activated ester of PEG-bis(succinimidyl succinate) was added very slowly with stirring. S-nitrosylation of PEG-Hb was then performed with addition of S-nitrosoglutathione. The yield of S-nitrosylation was estimated by using a high-performance liquid chromatography (HPLC) coupled with flow reactors of metal and Griess reagent (Fig. 2)6. In human Hb, is

Activated PEG Prydoxal 5'-phosphate (MW 3000 da)

Pyridoxalation ^

Pegylation S-Nitrosylation

Figure 1. SNO-PEG-Hb preparation. Hb was pyridoxalated, pegylated and then S-nitrosylated, highly reactive and the prefered target for S-nitrosylation. The content of NO in SNO-Hb reported in the text was, therefore, expressed on the basis that a fully s-nitrosylated Hb (100% SNO-Hb) contains two NOs because one tetramic Hb is constituted from two ß-subunits. The yield of S-nitrosylation was usually set to 30-37%.

A. SNO determination B. Heme determination

A. SNO determination B. Heme determination

Retention lime (min) Retention time (mtn)

Figure 2 HPLC characterization of (A) Hb-bound NO and (B) heme of SNO-PEG-Hb. (A) Samples were separated on a gel-filtration column (8 x 300 mm, GFC-200, Eicom, Kyoto, Japan) eluted with 10 mM acetate buffer, 0.1 mM EDTA, 100 mM sodium chloride, pH 5.5, at the flow rate of 0.55 mL/min. The eluate was mixed with 1.75 mM mercury chloride at the flow rate of 0.20 mL/min to decompose S-nitrosylated protein, and further mixed with Griess reagent at the flow rate of 0.22 mL/min. The red azo-dye formed was determined by the absorption at 540 nm. (B) For the characterization of molecular weight distribution, proteins were separated on a gelfiltration column (7.6 x 300 mm, TSK G3000SW, Toyo Soda Co. Ltd, Tokyo, Japan) in 10 mM sodium phosphate buffer, 100 mM sodium chloride, pH 6.9, at the flow rate of 0.9 mL/min. Proteins were monitored at 420 nm for heme and at 280 nm for molecular weight markers.

Retention lime (min) Retention time (mtn)

Figure 2 HPLC characterization of (A) Hb-bound NO and (B) heme of SNO-PEG-Hb. (A) Samples were separated on a gel-filtration column (8 x 300 mm, GFC-200, Eicom, Kyoto, Japan) eluted with 10 mM acetate buffer, 0.1 mM EDTA, 100 mM sodium chloride, pH 5.5, at the flow rate of 0.55 mL/min. The eluate was mixed with 1.75 mM mercury chloride at the flow rate of 0.20 mL/min to decompose S-nitrosylated protein, and further mixed with Griess reagent at the flow rate of 0.22 mL/min. The red azo-dye formed was determined by the absorption at 540 nm. (B) For the characterization of molecular weight distribution, proteins were separated on a gelfiltration column (7.6 x 300 mm, TSK G3000SW, Toyo Soda Co. Ltd, Tokyo, Japan) in 10 mM sodium phosphate buffer, 100 mM sodium chloride, pH 6.9, at the flow rate of 0.9 mL/min. Proteins were monitored at 420 nm for heme and at 280 nm for molecular weight markers.

PEG-Hb

Figure 3 Changes in mean arterial blood pressure 5 min after a bolus injection of Hb materials (125 mg Hb/kg, Hb 10% solution) into male Wistar rats. Relative increase was calculated from the data before and after the injection. Each value represents the mean±SEM of 6-9 animals. *p<0.05 vs saline control by ANOVA followed by Scheme's test.

PEG-Hb

Figure 3 Changes in mean arterial blood pressure 5 min after a bolus injection of Hb materials (125 mg Hb/kg, Hb 10% solution) into male Wistar rats. Relative increase was calculated from the data before and after the injection. Each value represents the mean±SEM of 6-9 animals. *p<0.05 vs saline control by ANOVA followed by Scheme's test.

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