Properties of carboxymethylated cross-linked hemoglobin A.
The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)
Fantl
W J
WJ
Rockefeller University, New York, New York 10021.
Manning
L R
LR
Ueno
H
H
Di Donato
A
A
Manning
J M
JM
D000079:Acetaldehyde / Q000031:analogs & derivatives; D000596:Amino Acids / Q000032:analysis; D003432:Cross-Linking Reagents; D006441:Hemoglobin A / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D010108:Oxyhemoglobins / Q000378:metabolism
D000596:Amino Acids; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D010108:Oxyhemoglobins; D006441:Hemoglobin A; C029774:oxyhemoglobin A; D000079:Acetaldehyde; C010972:glycolaldehyde