(2,5-dioxopyrrolidin-1-yl) 6-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a sample bottle (8 mL volume), it was charged by 0.39 mL of human CO hemoglobin (Hb) solution (509 μM), and diluted with 1.61 mL of phosphate buffered saline solution (PBS) to be 0.1 mM 2 mL. Next, 7.6 mg of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH, manufactured by Pierce Corporation) was dissolved in 0.25 mL of dimethyl sulfoxide (DMSO) to prepare 80 mM dimethyl sulfoxide (DMSO) solution of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH). To the above human hemoglobin (Hb) solution (2 mL), it was added with 0.047 mL of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) solution (succinimidyl-6-(β-maleimidopropionamido)hexanoate/human hemoglobin (SMPH/Hb) =18 (mol/mol)), while stirring (100 rpm), and stirred at room temperature for 30 minutes. From the resultant reaction solution, unreacted succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) was removed by the same method as the method described in Preparation Example 2 in Example 1. Specifically, 2.1 mL of human hemoglobin (Hb) solution was moved to a centrifugal concentrator (Vivaspin 20), diluted with about 18 mL of phosphate buffered saline solution (PBS) to ten times, and then concentrated to about 1.0 mL ( 1/10) at 4000 rpm, for 30 minutes and at 4° C. To that, it was added with 19 mL of phosphate buffered saline solution (PBS), and concentrated to about 1.0 mL under the same conditions. By repeating this dilution/concentration operation three times, unreacted succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) was removed. Lastly, a sample in a tube was moved to a sample bottle (8 mL volume), and the total volume was adjusted with phosphate buffered saline solution (PBS) to 2.0 mL. The human hemoglobin-crosslinker conjugate (Hb-SMPH) concentration became 0.1 mM. stirred at room temperature for 30 minutes was removed by the same method as the method described in Preparation Example 2 in Example 1 concentrated to about 1.0 mL ( 1/10) at 4000 rpm, for 30 minutes and at 4° C To that, it was added with 19 mL of phosphate buffered saline solution (PBS) concentrated to about 1.0 mL under the same conditions was removed Lastly, a sample in a tube was moved to a sample bottle (8 mL volume) The human hemoglobin-crosslinker conjugate (Hb-SMPH) concentration

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a sample bottle (8 mL volume), it was charged by 0.39 mL of human CO hemoglobin (Hb) solution (509 μM), and diluted with 1.61 mL of phosphate buffered saline solution (PBS) to be 0.1 mM 2 mL. Next, 7.6 mg of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH, manufactured by Pierce Corporation) was dissolved in 0.25 mL of dimethyl sulfoxide (DMSO) to prepare 80 mM dimethyl sulfoxide (DMSO) solution of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH). To the above human hemoglobin (Hb) solution (2 mL), it was added with 0.047 mL of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) solution (succinimidyl-6-(β-maleimidopropionamido)hexanoate/human hemoglobin (SMPH/Hb) =18 (mol/mol)), while stirring (100 rpm), and stirred at room temperature for 30 minutes. From the resultant reaction solution, unreacted succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) was removed by the same method as the method described in Preparation Example 2 in Example 1. Specifically, 2.1 mL of human hemoglobin (Hb) solution was moved to a centrifugal concentrator (Vivaspin 20), diluted with about 18 mL of phosphate buffered saline solution (PBS) to ten times, and then concentrated to about 1.0 mL ( 1/10) at 4000 rpm, for 30 minutes and at 4° C. To that, it was added with 19 mL of phosphate buffered saline solution (PBS), and concentrated to about 1.0 mL under the same conditions. By repeating this dilution/concentration operation three times, unreacted succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) was removed. Lastly, a sample in a tube was moved to a sample bottle (8 mL volume), and the total volume was adjusted with phosphate buffered saline solution (PBS) to 2.0 mL. The human hemoglobin-crosslinker conjugate (Hb-SMPH) concentration became 0.1 mM. stirred at room temperature for 30 minutes was removed by the same method as the method described in Preparation Example 2 in Example 1 concentrated to about 1.0 mL ( 1/10) at 4000 rpm, for 30 minutes and at 4° C To that, it was added with 19 mL of phosphate buffered saline solution (PBS) concentrated to about 1.0 mL under the same conditions was removed Lastly, a sample in a tube was moved to a sample bottle (8 mL volume) The human hemoglobin-crosslinker conjugate (Hb-SMPH) concentration

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a sample bottle (8 mL volume), it was charged by 0.39 mL of human CO hemoglobin (Hb) solution (509 μM), and diluted with 1.61 mL of phosphate buffered saline solution (PBS) to be 0.1 mM 2 mL. Next, 7.6 mg of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH, manufactured by Pierce Corporation) was dissolved in 0.25 mL of dimethyl sulfoxide (DMSO) to prepare 80 mM dimethyl sulfoxide (DMSO) solution of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH). To the above human hemoglobin (Hb) solution (2 mL), it was added with 0.047 mL of succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) solution (succinimidyl-6-(β-maleimidopropionamido)hexanoate/human hemoglobin (SMPH/Hb) =18 (mol/mol)), while stirring (100 rpm), and stirred at room temperature for 30 minutes. From the resultant reaction solution, unreacted succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) was removed by the same method as the method described in Preparation Example 2 in Example 1. Specifically, 2.1 mL of human hemoglobin (Hb) solution was moved to a centrifugal concentrator (Vivaspin 20), diluted with about 18 mL of phosphate buffered saline solution (PBS) to ten times, and then concentrated to about 1.0 mL ( 1/10) at 4000 rpm, for 30 minutes and at 4° C. To that, it was added with 19 mL of phosphate buffered saline solution (PBS), and concentrated to about 1.0 mL under the same conditions. By repeating this dilution/concentration operation three times, unreacted succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH) was removed. Lastly, a sample in a tube was moved to a sample bottle (8 mL volume), and the total volume was adjusted with phosphate buffered saline solution (PBS) to 2.0 mL. The human hemoglobin-crosslinker conjugate (Hb-SMPH) concentration became 0.1 mM. stirred at room temperature for 30 minutes was removed by the same method as the method described in Preparation Example 2 in Example 1 concentrated to about 1.0 mL ( 1/10) at 4000 rpm, for 30 minutes and at 4° C To that, it was added with 19 mL of phosphate buffered saline solution (PBS) concentrated to about 1.0 mL under the same conditions was removed Lastly, a sample in a tube was moved to a sample bottle (8 mL volume) The human hemoglobin-crosslinker conjugate (Hb-SMPH) concentration