Relationship between protein/solvent proton exchange and progressive conformation and fluctuation changes in hemoglobin

Abstract

The pH dependence of the tertiary structural changes and the quaternary reorganization of the a j interface of oxyhemoglobin in solution has been previously observed in our laboratory. The present work aims to establish whether or not these progressive structural changes with pH result from proton exchange between the protein and the solvent. We therefore have used infrared spectroscopy, acidlbase titration and 'HI2H exchange to assess the effect of external proton concentration on the structural and dynamic properties of the hemoglobin molecule in solution. This study is also performed on the carbonmonoxy form since the affinity of hemoglobin for CO is much higher than for oxygen. The present findings demonstrate that affinity changes arc closely related to protein fluctuations, as well as structural modifications. An increased affinity, induced either by ligand replacement or pH variation, is associated with the constrained chains exhibiting a lower degree of fluctuation, together with a looser a/3 interface association It was recently shown that the tertiary and quaternary structures of oxyhemoglobin in solution evolve continuously with pH [l]. This gradual development was probed by observing the SH stretching vibration of two cysteinyl residues: aCysl04 (G11) and /3Cysll2 (G14). These two cysteine residues are known to be strongly H-bonded and very sensitive to slight conformational changes which are undetectable by other techniques [2, 31. The H-bond affects the distance between the sulfur and hydrogen atoms and, consequently, perturbs the SH vibration. The aCysl04 (G11) residue provides detailed information on the tertiary constraints of the G chain a-helices whereas the thiol group of pCysll2 (G14) is sensitive to quaternary structural changes at the tight ap interface Although the functional properties of hemoglobin are known to be pH dependent [5 -81, the changes in interactions inside the protein, induced by the surrounding solvent, are not yet clearly understood. While the affinity of hemoglobin for oxygen increases with pH, the molecular constraints at the x p interface decrease and the intrachain H-bonds of the a chain become stronger [I]. To investigate further the effect of pH on the molecular organization of hemoglobin, the Fouriertransform infrared study of the stretching vibrations of SH was extended to the carbonmonoxy hemoglobin derivative since the partition coefficient between O2 and CO is about 260 for the fully ligated hemoglobin [7]. Furthermore, in order to obtain a more general understanding of the mechanisms correlating the proton concentration in the external solvent with the dynamic properties of these two hemoglobin forms (0, and CO), we have studied their acid/base titration behaviour and their 'H/2H exchange variations with pH. Indeed, many protons of the protein are exchangeable with those of the external solvent [9 -121. Besides, Lumry and Rosenberg

Hemoglobin Dynamics in Red Blood Cells: Correlation to Body Temperature

“…At temperatures .36.9°C, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence. These results indicate partial unfolding of hemoglobin at around human body temperature, which is in agreement with the results of CD experiments (1,3,47). It was deduced that these changes are caused mostly by amino acid side chains toward the exterior and on the surface of the proteins.…”

“…Although incoherent scattering is predominantly due to nonexchangeable hydrogen atoms of the protein, scattering from D 2 O solvent contributes partially to the signal. The incoherent scattering cross section of human carbonmonoxy hemoglobin was estimated from the amino acid sequence taken from pdb file 2DN3 (46), assuming that 13% of the protons exchange with deuterons (47). The fraction of D 2 O in the sample was determined by drying and weighting of an aliquot.…”

Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection

“…All the protein solutions were centrifuged at 14 000 g for 5 min at 4 °C before use. Protein concentrations were measured on a Shimadzu UV-2450 spectrophotometer. , Hemoglobin integrity was checked by measuring the absorption coefficient ratio ε 576 /ε 541 indicative of iron oxidation and protein damage . Hemoglobin concentration is expressed as heme molar concentration.…”

“…38,39 Hemoglobin integrity was checked by measuring the absorption coefficient ratio ε 576 /ε 541 indicative of iron oxidation and protein damage. 40 Hemoglobin concentration is expressed as heme molar concentration.…”

<title>Near-infrared spectroscopy of lysed blood: pH effects</title>

“…Titration data available for albumin, the largest constituent in plasma, confirm that over the [5][6][7][8] pH range very little buffering capacity is present.16…”