PMID- 10529219
OWN - NLM
STAT- MEDLINE
DCOM- 19991110
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 40
DP  - 1999 Oct 5
TI  - Assessment of roles of surface histidyl residues in the molecular basis of the
      Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate
      in human normal adult hemoglobin.
PG  - 13423-32
AB  - Site-directed mutagenesis has been used to construct two mutant recombinant
      hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to
      assign the C2 proton resonances of beta116His and beta143His and to resolve the
      ambiguous assignments made over the past years. In the present work, we have
      identified the C2 proton resonances of two surface histidyl residues of the beta 
      chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by
      comparing the proton nuclear magnetic resonance (NMR) spectra of human normal
      adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other
      previous assignments complete the assignments for all 24 surface histidyl
      residues of human normal adult hemoglobin. The individual pK values of 24
      histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M 
      N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the
      presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2
      proton resonances of the histidyl residues as a function of pH. Among those
      surface histidyl residues, beta146His has the biggest contribution to the
      alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution
      to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His 
      have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, 
      and beta116His have moderate positive contributions; and beta2His and beta77His
      have a moderate negative contribution to the Bohr effect. The sum of the
      contributions from 24 surface histidyl residues accounted for 86% of the alkaline
      Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although
      beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG)
      according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG,
      beta143His is not essential for the binding of 2,3-BPG in the neutral pH range
      according to the proton NMR and oxygen affinity studies presented here. With the 
      accurately measured and assigned individual pK values for all surface histidyl
      residues, it is now possible to evaluate the Bohr effect microscopically for
      novel recombinant Hbs with important functional properties, such as low oxygen
      affinity and high cooperativity. The present study further confirms the
      importance of a global electrostatic network in regulating the Bohr effect of the
      hemoglobin molecule.
FAU - Fang, T Y
AU  - Fang TY
AD  - Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,
      Pennsylvania 15213, USA.
FAU - Zou, M
AU  - Zou M
FAU - Simplaceanu, V
AU  - Simplaceanu V
FAU - Ho, N T
AU  - Ho NT
FAU - Ho, C
AU  - Ho C
LA  - eng
GR  - HL-24525/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Hemoglobins)
RN  - 0 (Peptide Fragments)
RN  - 0 (Protons)
RN  - 0 (Recombinant Proteins)
RN  - 138-81-8 (2,3-Diphosphoglycerate)
RN  - 4QD397987E (Histidine)
RN  - 9008-02-0 (deoxyhemoglobin)
RN  - 9034-51-9 (Hemoglobin A)
RN  - 9034-63-3 (Fetal Hemoglobin)
RN  - 9061-29-4 (Carboxyhemoglobin)
RN  - J65BV539M3 (Deuterium Oxide)
RN  - S88TT14065 (Oxygen)
SB  - IM
MH  - 2,3-Diphosphoglycerate/blood/*chemistry
MH  - Adult
MH  - Binding Sites/genetics
MH  - Carboxyhemoglobin/chemistry/metabolism
MH  - Deuterium Oxide/chemistry
MH  - Fetal Hemoglobin/chemistry/metabolism
MH  - Hemoglobin A/chemistry/metabolism
MH  - Hemoglobins/*chemistry/genetics/metabolism
MH  - Histidine/*chemistry
MH  - Humans
MH  - Hydrogen-Ion Concentration
MH  - Mass Spectrometry
MH  - Mutagenesis, Site-Directed
MH  - Nuclear Magnetic Resonance, Biomolecular
MH  - Oxygen/*blood
MH  - Peptide Fragments/chemistry/genetics/metabolism
MH  - Protons
MH  - Recombinant Proteins/chemistry/isolation & purification/metabolism
EDAT- 1999/10/21 00:00
MHDA- 1999/10/21 00:01
CRDT- 1999/10/21 00:00
PHST- 1999/10/21 00:00 [pubmed]
PHST- 1999/10/21 00:01 [medline]
PHST- 1999/10/21 00:00 [entrez]
AID - bi9911379 [pii]
AID - 10.1021/bi9911379 [doi]
PST - ppublish
SO  - Biochemistry. 1999 Oct 5;38(40):13423-32. doi: 10.1021/bi9911379.