Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications.
The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.
Kerwin
B A
BA
Amgen, One Amgen Center Drive, Mail Drop 8-1-C, Thousand Oaks, California 91320-1799, USA. bkerwin@amgen.com
Akers
M J
MJ
Apostol
I
I
Moore-Einsel
C
C
Etter
J E
JE
Hess
E
E
Lippincott
J
J
Levine
J
J
Mathews
A J
AJ
Revilla-Sharp
P
P
Schubert
R
R
Looker
D L
DL
D001205:Ascorbic Acid / Q000737:chemistry*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D010434:Pepsin A / Q000737:chemistry; D010449:Peptide Mapping; D011136:Polysorbates; D012542:Scattering, Radiation; D012996:Solutions; D014357:Trypsin / Q000737:chemistry
D005296:Ferrous Compounds; D006454:Hemoglobins; D011136:Polysorbates; D012996:Solutions; C100003:deoxyhemoglobin; D014357:Trypsin; D010434:Pepsin A; D001205:Ascorbic Acid; D010100:Oxygen