High-resolution 31P Nuclear Magnetic Resonance Research Articles

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Plasmalogen loss caused by remodeling deficiency in mitochondria.

Lipid homeostasis is crucial in human health. Barth syndrome (BTHS), a life-threatening disease typically diagnosed with cardiomyopathy and neutropenia, is caused by mutations in the mitochondrial transacylase tafazzin. By high-resolution 31P nuclear magnetic resonance (NMR) with cryoprobe technology, recently we found a dramatic loss of choline plasmalogen in the tafazzin-knockdown (TAZ-KD) mouse heart, besides observing characteristic cardiolipin (CL) alterations in BTHS. In inner mitochondrial membrane where tafazzin locates, CL and diacyl phosphatidylethanolamine are known to be essential via lipid-protein interactions reflecting their cone shape for integrity of respiratory chain supercomplexes and cristae ultrastructure. Here, we investigate the TAZ-KD brain, liver, kidney, and lymphoblast from patients compared with controls. We identified common yet markedly cell type-dependent losses of ethanolamine plasmalogen as the dominant plasmalogen class therein. Tafazzin function thus critically relates to homeostasis of plasmalogen, which in the ethanolamine class has conceivably analogous and more potent molecular functions in mitochondria than diacyl phosphatidylethanolamine. The present discussion of a loss of plasmalogen-protein interaction applies to other diseases with mitochondrial plasmalogen loss and aberrant forms of this organelle, including Alzheimer's disease.

A DOPO based reactive flame retardant constructed by multiple heteroaromatic groups and its application on epoxy resin: curing behavior, thermal degradation and flame retardancy

A high-efficiency flame retardant composed of phosphaphenanthrene, benzothiazole and imidazole groups (PBI) was synthesized and served as flame retardant co-curing agent to reduce the fire hazard of epoxy resin (EP). The chemical structure of PBI was characterized by Fourier transform infrared spectroscopy (FTIR), high-resolution mass spectroscopy (HR-MS), 1H and 31P nuclear magnetic resonance (NMR). The curing behavior, thermal stability and flame retardant properties of the prepared EP systems were investigated. The curing behavior study disclosed that PBI accelerated the crosslinking reaction of EP and was chemically bonded with EP matrix to obtain intrinsic flame retardant thermoset. The resulting EP thermosets showed only slight decrease in glass transition temperature (Tg). The thermogravimetric analysis (TGA) results indicated both the catalytic decomposition and catalytic charring effects of PBI demonstrated by decreased thermal stability and enhanced charring capability of EP/DDS/PBI thermosets. The combustion test results showed remarkable improvement in the flame retardant properties of EP/DDS/PBI thermosets. When the phosphorus content was only 0.75 wt%, EP/DDS/PBI-0.75 thermoset achieved a limiting oxygen index (LOI) value of 34.6% and passed UL94 V-0 rating. Moreover, the peak of heat release rate (pk-HRR), average of heat release rate (av-HRR) and total heat release (THR) values were decreased by 48.7%, 31.1% and 28.3%, respectively, in comparison with those of the EP/DDS thermoset. The flame retardant effect of PBI on EP was attributed to the catalytic charring effect to form protective char layer in condensed phase and release of free radicals with quenching effect and nonflammable gases with diluting effect in gaseous phase.

Substantial Decrease in Plasmalogen in the Heart Associated with Tafazzin Deficiency.

Tafazzin is the mitochondrial enzyme that catalyzes transacylation between a phospholipid and a lysophospholipid in remodeling. Mutations in tafazzin cause Barth syndrome, a potentially life-threatening disease with the major symptom being cardiomyopathy. In the tafazzin-deficient heart, cardiolipin (CL) acyl chains become abnormally heterogeneous unlike those in the normal heart with a single dominant linoleoyl species, tetralinoleoyl CL. In addition, the amount of CL decreases and monolysocardiolipin (MLCL) accumulates. Here we determine using high-resolution 31P nuclear magnetic resonance with cryoprobe technology the fundamental phospholipid composition, including the major but oxidation-labile plasmalogens, in the tafazzin-knockdown (TAZ-KD) mouse heart as a model of Barth syndrome. In addition to confirming a lower level of CL (6.4 ± 0.1 → 2.0 ± 0.4 mol % of the total phospholipid) and accumulation of MLCL (not detected → 3.3 ± 0.5 mol %) in the TAZ-KD, we found a substantial reduction in the level of plasmenylcholine (30.8 ± 2.8 → 18.1 ± 3.1 mol %), the most abundant phospholipid in the control wild type. A quantitative Western blot revealed that while the level of peroxisomes, where early steps of plasmalogen synthesis take place, was normal in the TAZ-KD model, expression of Far1 as a rate-determining enzyme in plasmalogen synthesis was dramatically upregulated by 8.3 (±1.6)-fold to accelerate the synthesis in response to the reduced level of plasmalogen. We confirmed lyso-plasmenylcholine or plasmenylcholine is a substrate of purified tafazzin for transacylation with CL or MLCL, respectively. Our results suggest that plasmenylcholine, abundant in linoleoyl species, is important in remodeling CL in the heart. Tafazzin deficiency thus has a major impact on the cardiac plasmenylcholine level and thereby its functions.

Alteration of phospholipids in the blood of patients with Duchenne muscular dystrophy (DMD): in vitro, high resolution 31P NMR-based study.

In vitro, high-resolution 31P NMR (Nuclear Magnetic Resonance) spectroscopy-based analysis of phospholipids in serum is well recognized in leukemia, lymphoma, non-hematological cancers and renal cell carcinoma. In context of these studies, phospholipids were analyzed in blood of thirty-two (n=32) patients with Duchenne muscular dystrophy (DMD) (Age, Mean±SD; 8.0±1.6years) and sixteen (n=16) healthy subjects (Age, Mean±SD; 8.6±2.3years). Quantity of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and lyso-phosphatidylcholine (Lys-PC) was significantly higher (p<0.05) in DMD patients as compared to healthy subjects. There were no significant differences (p>0.05) observed for the quantity of phospholipids in blood of gene deletion positive cases of DMD as compared to negative gene deletion cases of DMD. Quantity of phospholipids in negative gene deletion cases of DMD patients as well as DMD cases with positive gene deletion was significantly higher (p<0.05) as compared to normal individuals. The present study distinguishes the patients with DMD from the healthy subjects on the basis of the quantity of phospholipids in blood. These observations may be useful in future for the development of new diagnostic method of DMD.

31P nuclear magnetic resonance study of the proton-irradiated KTiOPO4

31P nuclear magnetic resonance (NMR) was employed to study the effects of proton irradiation on KTiOPO4 (KTP) in view of the previously studied paramagnetic impurity doping effects. High-resolution 31P NMR measurements showed significant increase in the isotropic chemical shifts of the two inequivalent phosphorus sites in the proton-irradiated KTP system, indicating decrease in the electron density around the phosphorous nuclei. The 31P NMR linewidths of the KTP system manifested anomalies associated with the superionic transition and with the polaron formation, which became much weaker after proton irradiation. Besides, the activation energy of the charge carriers increased significantly after proton irradiation.

NMR and X-ray diffraction study of the phases of zirconium phosphate incorporated in a composite membrane Nafion®-ZrP

Zirconium Phosphate (ZrP) inside a composite membrane Nafion-ZrP was studied by combining wide angle X-ray diffraction (XRD) and high-resolution solid-state 31P Nuclear Magnetic Resonance (NMR) under magic angle spinning conditions (MAS). We have, at first, investigated the physical features of ZrP powders that were prepared ex situ under different conditions leading to either amorphous or crystalline α-ZrP. ZrP incorporated into Nafion® membranes (in situ ZrP) was then studied. Informations obtained from X-ray and NMR data clearly show that the in situ ZrP is not in a purely crystalline phase. The presence of broad peaks in the X-ray spectra proves that the compound inside the membrane is semi-crystalline. This result is confirmed by MAS 31P NMR since the NMR spectra of in situ ZrP and ex situ semi-crystalline ZrP powder are alike. Moreover, the NMR analyses demonstrate that the phosphate groups can be found in three different connectivities with phosphorous atoms surrounded by either 2, 3 or 4 OZr bonds., with predominantly 3 OZr bonds being favored.

Coupling between the protons and PO4 tetrahedra in H+-ion-irradiated TlH2PO4

We studied hydrogen-bonded TlH2PO4 (TDP) ferroelectrics treated with proton-beam bombardment. The TDP material was irradiated with a 1-MeV proton beam at a dose of 1015/cm2. To analyze the local microscopic structure of TDP, we conducted 1H combined rotation and multiple pulse spectroscopy (CRAMPS), nuclear magnetic resonance (NMR), and high-resolution 31P NMR measurements. The isotropic chemical shifts of hydrogen and phosphor nuclei exhibited displacive features throughout the antiferroelectric, ferroelastic, and paraelastic phases that were attributed to the interplay of PO4 lattice deformation and the microscopic hydrogen bond network. The temperature dependence of the isotropic chemical shift σiso in 1H and high resolution 31P NMR revealed that the electronic charge redistribution was induced by proton-beam irradiation and the material’s elastic properties. We examined the proton-lattice relationship in terms of the proton’s location in a local double minimum well and the displacive character of the PO4 lattice.

Phospholipid compositions of sera and synovial fluids from dog, human and horse: a comparison by 31P‐NMR and MALDI‐TOF MS

Alterations of the phospholipid (PL) compositions of body fluids are assumed to be indicative of inflammatory diseases, e.g. rheumatoid arthritis (RA). Recently, we have shown that particularly the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio determined in human synovial fluids (SF) and sera represents a reliable measure of the inflammatory state in RA patients. However, it is not yet clear to what extent the PC/LPC ratio is also affected by nutrition habits. In the present study, the PL and the corresponding acyl chain compositions of human body fluids (SF and serum of RA patients as well as serum from healthy volunteers) are compared with those of two other mammalian species (horses and dogs suffering from degenerative joint diseases as well as healthy controls) by high-resolution 31P-nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). The most important result of this study is that the PL compositions of SF and serum of horse and dog are comparable with those of human body fluids. Compared with humans, however, the horse body fluid contains less PCs with highly unsaturated arachidonoyl residues, while that of dogs possesses the highest content of arachidonoyl-containing PC. These species-related differences stem primarily from different nutrition habits (meat vs. plants).

NMR and impedance study of H+-ion irradiated TlH2PO4

ABSTRACTThe H+-ion treatment effect on TlH2PO4, a KH2PO4 (KDP)-type ferroelectrics, was studied by nuclear magnetic resonance (NMR) and AC dielectric measurements. A sample of TlH2PO4 was irradiated by 1-MeV H+ ion beams to a dose of 1015 ions/cm2. The irradiation changed the hydrogen-bond geometry, presumably affecting the order-disorder proton dynamics. The deformation of the PO4 tetrahedra was identified by the isotropic chemical shift and the full width at half maximum (FWHM) of the high-resolution 31P NMR spectra. A prominent decrease in the dielectric constant was also observed after the irradiation. The macroscopic and microscopic changes due to the irradiation are discussed in the light of the proton dynamics.

High-resolution 31P nuclear magnetic resonance study of phase transitions in TlH2PO4

Abstract High-resolution 31 P nuclear magnetic resonance (NMR) techniques were employed to study a KH 2 PO 4 -type ferroelectric system, TlH 2 PO 4 . A marked temperature dependence of the isotropic chemical shift below the ferroelastic phase transition temperature is indicative of an electronic instability. The NMR linewidth showed a discontinuity at the ferroelastic phase transition, and the anisotropy was measured to increase rapidly below the antiferroelectric phase transition. Thus, the changes in the microscopic environments associated with the phase transitions were sensitively reflected in a characteristic manner.

Asymmetrical Membranes and Surface Tension

The 31P-nuclear magnetic resonance chemical shift of phosphatidic acid in a membrane is sensitive to the lipid head group packing and can report qualitatively on membrane lateral compression near the aqueous interface. We have used high-resolution 31P-nuclear magnetic resonance to evaluate the lateral compression on each side of asymmetrical lipid vesicles. When monooleoylphosphatidylcholine was added to the external monolayer of sonicated vesicles containing dioleoylphosphatidylcholine and dioleoylphosphatidic acid, the variation of 31P chemical shift of phosphatidic acid indicated a lateral compression in the external monolayer. Simultaneously, a slight dilation was observed in the inner monolayer. In large unilamellar vesicles on the other hand the lateral pressure increased in both monolayers after asymmetrical insertion of monooleoylphosphatidylcholine. This can be explained by assuming that when monooleoylphosphatidylcholine is added to large unilamellar vesicles, the membrane bends until the strain is the same in both monolayers. In the case of sonicated vesicles, a change of curvature is not possible, and therefore differential packing in the two layers remains. We infer that a variation of lipid asymmetry by generating a lateral strain in the membrane can be a physiological way of modulating the conformation of membrane proteins.

Phospholipid abnormalities in postmortem schizophrenic brains detected by 31P nuclear magnetic resonance spectroscopy: a preliminary study

It has been hypothesized that schizophrenia arises from cell membrane abnormalities due to changes in phospholipid (PL) composition and metabolism. We have used high resolution, in vitro 31P nuclear magnetic resonance (NMR) to characterize the PLs in left frontal cortex (gray matter) of postmortem brain from four schizophrenics and five controls. High resolution 31P NMR spectra were obtained in an organic-solvent system to resolve PL classes (headgroups) and in a sodium-cholate, aqueous dispersion system to resolve phosphatidylcholine (PC) molecular species. Multivariate analysis which included the major PC molecular species and phosphatidylinositol (PI) showed a significant difference between schizophrenics and controls. Analysis of specific interactions showed that the PI was significantly higher in the schizophrenic group than in the control group. There were no differences between the two groups for other individual PL classes, or for individual PL subclasses determined by the linkage type at the sn-1 position on glycerol. There was a trend for total PL content to be higher in schizophrenics than in controls. There was no evidence for elevated lysophosphatidylcholine or lysophosphatidylethanolamine in schizophrenia. The intensity of the PC peak representing molecular species with one saturated and one unsaturated (one or two double bonds) acyl chain was higher for the schizophrenic group than for the control group. Although these results are not in complete agreement with previous studies, they support the idea that PL abnormalities occur in the brain in schizophrenia and that fatty acid metabolism may be abnormal.

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G 8 and A 9, a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the 31P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. 31P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S–substrate complexes generated in the absence of Mg 2+ ions were still in an inactive form and that Mg 2+ ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.

Perturbations of glucose metabolism associated with HIV infection in human intestinal epithelial cells: a multinuclear magnetic resonance spectroscopy study.

To analyse the effect of HIV-1 infection on the glucose metabolism of human intestinal epithelial cells. HT-29 cells were infected with HIV-1NDK and studied 3 weeks (acutely infected cells) or 9 months (chronically infected cells) post-infection. Perchloric acid extracts were analysed by high-resolution 1H, 31P and 13C nuclear magnetic resonance spectroscopy. Metabolite concentrations and specific 13C enrichments were quantified for chronically infected, acutely infected and control cells grown in Dulbecco's modified Eagle's medium containing natural-abundance or 1-13C-enriched glucose to determine significant differences between infected and non-infected cells. Chronically HIV-infected cells showed alterations in glycerol-3-phosphate (+40%), fructose-1,6-diphosphate (-66%), uridine diphosphate glucuronic acid (-33%), lactate (+75%) and [1-13C]glucose (+181%) levels, and in specific lactate 3-13C enrichment (+19%) when compared with controls. Acutely infected cells exhibited decreased fructose-1,6-diphosphate (-58%) and increased nicotinamide adenine dinucleotide (+33%) levels relative to controls. HIV-1 infection results in a disturbance of glycolytic and oxidative activities in human intestinal epithelial cells. This finding supports the concept that HIV-1 may directly impair some metabolic functions of the intestinal epithelium, and that it can be considered a potential aetiological agent for HIV-associated enteropathy.

Production and release of polyphosphate by a genetically engineered strain of Escherichia coli

A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium. PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis. PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium. When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed. Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C. These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E. coli recombinants. High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E. coli recombinant. The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium. Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.

Binding of germanium and lead to Pseudomonas putida lipopolysaccharides

The binding of germanium and lead ions to lipopolysaccharide isolated from Pseudomonas putida ATCC 33015 was studied by high-resolution 31P nuclear magnetic resonance spectroscopy. Chemical shift changes toward higher field as well as broadening of all phosphorus resonances were observed with added lead. Addition of germanium to the lipopolysaccharide sample resulted in more pronounced chemical shift changes toward lower field. The effects of binding of germanium and lead ions on the 31P nuclear magnetic resonance spectrum of lipopolysaccharide imply that the glycosidic monophosphate joined to the reducing end of N-acetylglucosamine (C-1 position of α-D-G1cNI) as well as β-phosphate groups of glycosidically linked diphosphates of the same residue of the lipid A moiety provide high-affinity metal-binding sites.Key words: germanium, lead, lipopolisaccharide (LPS), binding, Pseudomonas putida.

Phosphocreatine hydrolysis by 31P-NMR at the onset of constant-load exercise in humans.

The kinetics of phosphocreatine (PC) breakdown in human plantar flexors at the onset of constant-load aerobic exercise was determined by high-resolution 31P-nuclear magnetic resonance spectroscopy (NMRS). The half time of the process (t1/2PC) was obtained by fitting curves (n = 13) from five subjects at various aerobic work loads for which muscle pH was not different from that at rest. Steady-state PC concentration ([PC]) was not < 70% of the resting value and was linearly related to the work load (w) ([PC] = -3.01 +/- 0.08 w + 1 (r = 0.48, 2P < 0.1)). The average t1/2PC was 16.2 s and was independent of work load. Because the half time of the muscle PC kinetics reflects the half time of the O2 uptake (MO2) kinetics (t1/2MO2), the latter is equal to that found earlier in the isolated perfused dog gastrocnemius. Whereas in the dog the above t1/2MO2 compares well with the homologous half time of the O2 uptake at the alveolar level, in humans such equivalence is found only at extremely low work loads, when the transient contribution by anaerobic glycolysis is negligible.

High-resolution solid-state 31P and 119Sn magic-angle spinning nuclear magnetic resonance studies of amorphous and microcrystalline layered metal(IV) hydrogenphosphates

High-resolution solid-state 31P and 119Sn magic-angle spinning nuclear magnetic resonance (MAS NMR) and Mössbauer spectroscopies have been used to probe the structure of amorphous and microcrystalline layered hydrogenphosphates and some of their intercalation compounds. A linear relationship between δ(31P) and the Allred–Rochow electronegativity of the metal atom was observed. In the case of both α and γ forms of titanium and zirconium hydrogenphosphates, a linear relationship was seen between the isotropic chemical shift (δiso) and the number of bridging P—O—M oxygens (connectivity) of the phosphate groups. In a further study of the intercalation compounds of tin(IV) hydrogenphosphate monohydrate (SnP), the NMR data indicated there to be more electron transfer in the propylamine intercalation compound than is the case with E-N,N′-diethylbut-2-ene-1,4-diamine (NNBD) and that there is more electron transfer in the case of NNBD than in the ammonium intercalation compound. A linear relationship was observed when δ(119Sn) was plotted as a function of δ(31P). The Sn Mössbauer spectrum of the host material was indicative of ionic character around the metal atom and similar to that of SnF4.

Proton and phosphorus-31 nuclear magnetic resonance investigation of the interaction between 2,3-diphosphoglycerate and human normal adult hemoglobin

High-resolution 1H and 31P nuclear magnetic resonance spectroscopy has been used to investigate the binding of 2,3-diphosphoglycerate to human normal adult hemoglobin and the molecular interactions involved in the allosteric effect of the 2,3-diphosphoglycerate molecule on hemoglobin. Individual hydrogen ion NMR titration curves have been obtained for 22-26 histidyl residues of hemoglobin and for each phosphate group of 2,3-diphosphoglycerate with hemoglobin in both the deoxy and carbonmonoxy forms. The results indicate that 2,3-diphosphoglycerate binds to deoxyhemoglobin at the central cavity between the two beta chains and the binding involves the beta 2-histidyl residues. Moreover, the results suggest that the binding site of 2,3-diphosphoglycerate to carbonmonoxyhemoglobin contains the same (or at least some of the same) amino acid residues responsible for binding in the deoxy form. As a result of the specific interactions with 2,3-diphosphoglycerate, the beta 2-histidyl residues make a significant contribution to the alkaline Bohr effect under these experimental conditions (up to 0.5 proton/Hb tetramer). 2,3-Diphosphoglycerate also affects the individual hydrogen ion equilibria of several histidyl residues located away from the binding site on the surface of the hemoglobin molecule, and, possibly, in the heme pockets. These results give the first experimental demonstration that long-range electrostatic and/or conformational effects of the binding could play an important role in the allosteric effect of 2,3-diphosphoglycerate on hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)