Influence of the chemical nature of side chain at beta 108 of hemoglobin A on the modulation of the oxygen affinity by chloride ions. Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: hemoglobins Presbyterian and Yoshizuka.
Hemoglobin A (HbA) and two low oxygen affinity variants of HbA, HbPresbyterian and HbYoshizuka, were produced in transgenic pigs and purified to homogeneity by ion-exchange chromatography. These two variants contain either lysine (HbPresbyterian) or aspartic acid (HbYoshizuka) instead of the normal asparagine residue at position beta 108 in HbA. Transgenic pigs expressed these variants at a level up to 11% and were healthy. Both HbPresbyterian and HbYoshizuka exhibited low O2 affinity (P50 of 21.2 and 18.9, respectively, compared with control HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooperativity with Hill coefficients of 2.9 and 2.5, respectively. HbPresbyterian exhibited Bohr effect comparable with HbA. In contrast, HbYoshizuka had a diminished response to changes in pH. Thus the structural basis of reduced O2 affinity of these variants appears to be distinct: the consequence of mutation at beta 108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O2 affinity of the variants to the presence of Cl-. The O2 affinity of HbYoshizuka is insensitive to changes in Cl- concentration, whereas the O2 affinity of HbPresbyterian exhibited a pronounced and dramatic chloride effect. In fact, P50 of HbPresbyterian was identical to that of HbA at very low Cl- concentrations, and the P50 increased to >40 at 0.5 M Cl-. The chloride effect was completely abolished when HbPresbyterian was stabilized at the 2,3-diphosphoglycerate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in HbPresbyterian, Cl- can bridge the epsilon-amino group of Lys beta 108 with either the guanidino group of Arg beta 104 or the epsilon-amino group of Lys alpha 99, resulting in the stabilization of the "T" structure. The utility of these low O2 affinity hemoglobins as cell-free oxygen carriers is discussed.
O'Donnell
J K
JK
DNX Biotherapeutics Inc., Princeton, New Jersey 08540.
Birch
P
P
Parsons
C T
CT
White
S P
SP
Okabe
J
J
Martin
M J
MJ
Adams
C
C
Sundarapandiyan
K
K
Manjula
B N
BN
Acharya
A S
AS
D000494:Allosteric Regulation; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry*; D003198:Computer Simulation; D017931:DNA Primers / Q000737:chemistry; D005914:Globins / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010108:Oxyhemoglobins / Q000737:chemistry*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013552:Swine
D002712:Chlorides; D017931:DNA Primers; D010108:Oxyhemoglobins; D005914:Globins; D006441:Hemoglobin A