ABSTRACT
In this study capillary zone electrophoresis (CZE) was used to analyze the poly(ethylene glycol)-modified bovine hemoglobin(PEG-bHb). The results show that CZE separated the subunit of bovine hemoglobin based on the number of PEG conjugating to the protein surface, which makes it possible to evaluate the degree of modification of hemoglobin subunit; meanwhile, it also reflected the stability of PEG attaching to hemoglobin after incubating with hydroxylamine, which makes it successful to detect the distribution of attachment site and evaluate the stability of PEG on the surface of hemoglobin. As a simple, fast and accurate method, CZE is suitable to monitor the production procedures and quality control of the final products of the PEG-bHb.
Subject(s)
Blood Substitutes/chemistry , Electrophoresis, Capillary/methods , Hemoglobins/chemistry , Polyethylene Glycols/chemistry , Animals , Blood Substitutes/chemical synthesis , Cattle , Drug Stability , Protein Subunits/chemistry , Quality ControlABSTRACT
To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.
