Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Chemical properties of the functional groups of insulin.

The method of competitive binding [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] with 1-fluoro-2,4-dinitrobenzene as the labelling reagent [Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175] was used to determine the chemical properties, namely pK and reactivity, of the amino groups, the histidine residues and the tyrosine residues of the dimeric form of pig zinc-free insulin at 20.0 degrees C. The N-terminal glycine residue of the A-chain has a pK of 7.7 and a slightly higher than normal reactivity. The N-terminal phenylalanine residue of the B-chain has a pK of 6.9 and is approximately an order of magnitude more reactive than a corresponding amino group with the same pK value. The lysine epsilon-amino group has an unusually low pK of 7.0 but has approximately the expected reactivity of such a group. In the case of the two histidine and four tyrosine residues only the average properties of each class were determined. The histidine residues have a pK value of approx. 6.6, but, however, their reactivity is at least an order of magnitude greater than that of a free imidazole group. The tyrosine residues have a pK value of approx. 10, but their average reactivities are substantially less than for a free phenolic group. At alkaline pH values above 8 the reactivity of all the functional groups show sharp discontinuities, indicating that insulin is undergoing a structural change that alters the properties of these groups.     

Locating active proton extrusion pumps in leaves

Abstract Stabilized microscopic preparations of an apoplastic fluorescent tracer, sulphorhodamine G (SR), have previously shown it confined to leaf cell walls. SR has a pK of 3.2, is dissociated at normal wall pH, and therefore does not enter cells. In transpiring soybean leaves, the SR showed a major internal water pathway in the walls of the paraveinal mesophyll (PVM), which has been implicated in the temporary storage of protein. Also the SR penetrated the PVM and bundle sheath cells, staining organelles and vacuoles, but not other leaf cells. This implies that sufficient SR is undissociated in these walls to allow penetration, and that the pH of the PVM walls is lower than that of most other cells. It is proposed that proton extrusion pumps are revealed by the low wall pH, and that these pumps are probably involved in collecting ammo acids from the transpiration stream.     

Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.     

Conformational differences between various myoglobin ligated states as monitored by 1H NMR spectroscopy

In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data

Electrostatic binding sites of extracellular polymeric substances (EPS) were characterized from titration data using linear programming analysis. Test results for three synthetic solutions of given solutes comprising amino, carboxyl, and phenolic groups indicated that this method was able to identify the electrostatic binding sites. For the six sites with pK(a) between 3 and 10, the estimated pK(a) deviated 0.11 +/- 0.09 from the theoretical values, and the estimated concentrations deviated 3.0% +/- 0.9% from the actual concentrations. Two EPS samples were then extracted from a hydrogen-producing sludge (HPS) and a sulfate-reducing biofilm (SRB). Analysis of charge excess data in titration from pH 3 to 11 indicated that the EPS of HPS comprised of five electrostatic binding sites with pK(a) ranging from 3 to 11. The pK(a) values of these binding sites and the possible corresponding functional groups were pK(a) 4.8 (carboxyl), pK(a) 6.0 (carboxyl/phosphoric), pK(a) 7.0 (phosphoric), pK(a) 9.8 (amine/phenolic), and pK(a) 11.0 (hydroxyl). EPS of the SRB comprised five of similar binding sites (with corresponding pK(a) values of 4.4, 6.0, 7.4, 9.4, and 11.0), plus one extra site at pK(a) 8.2, which was likely corresponding to the sulfhydryl group. The total electrostatic binding site concentration of EPS extracted from HPS were 10.88 mmol/g-EPS, of which the highest concentration was from the site of pK(a) 11.0. The corresponding values for the EPS extracted from SRB were 16.44 mmol/g-EPS and pK(a) 4.4. The total concentrations of electrostatic binding sites found in this study were 20- to 30-fold of those reported for bacterial cell surface, implying that EPS might be more crucial in biosorption of metals than bacterial cell surface in wastewater treatment and in bioremediation.     

pH-dependent photoisomerization of retinal in proteorhodopsin

The early steps in the photocycle of the bacterial proton pump proteorhodopsin (PR) were analyzed by ultrafast pump/probe spectroscopy to compare the rate of retinal isomerization at alkaline and acidic pH values. At pH 9, the functionally important primary proton acceptor (Asp97, pK(a) = 7.7) is negatively charged; consequently, a reaction cycle analogous to the archaeal bacteriorhodopsin (BR) is observed. The excited electronic state of PR displays a pronounced biphasic decay with time constants of 400 fs and 8 ps. At pH 6 where Asp97 is protonated a similar biphasic decay is observed, although it is significantly slower (700 fs and 15 ps). The results indicate, in agreement to similar findings in other retinal proteins, that also in PR the charge distribution within the chromophore binding pocket is a major determinant for the rate and the efficiency of the primary reaction.     

Reactivity of the active-centre lysine residue of rabbit muscle aldolase

The method of competitive labelling with [(3)H]acetic anhydride as the labelling reagent was used to determine the properties of the active-centre lysine residue of rabbit muscle aldolase. This residue is much less reactive than a normal exposed lysine residue towards this reagent, and its reactive properties did not parallel the pH-activity profile for aldolase. At higher pH values it became reactive, but this was shown to be due to disruption of the enzyme structure. The binding of the competitive inhibitor phosphate did not alter the reactive properties. It is concluded that the active-centre lysine has an apparent pK(a) greater than 11.5 and probably is made nucleophilic during the catalytic process, perhaps by proton abstraction.     

The fine structure of Herpetosiphon,and a note on the taxonomy of the genus

The fine structure of the Gram-negative filamentous gliding bacterium, Herpetosiphon is described. The outer membrane of the cell envelope could not be resolved as a separate structure, probably because it is fused with the underlying dense (peptidoglycan) layer. There was an additional wall layer outside this membrane-peptidoglycan complex, but a sheath in the classical sense, as postulated in the definition of the genus, was lacking. On the cell surface a loose network of fibrils could be seen. Inside the cells 3 types of intracytoplasmic membranes were discernible: a) true mesosomes near cross walls; b) a system of coarser membranes which was not connected with the septa and formed networks or tubular complexes; c) degenerated septa within bulbs. the bulbs are swollen sections of filaments, occurred mainly in ageing cultures, and are probably a degeneration phenomenon. The filaments contained necridia, i.e. dead and empty cells, across which breaks may occur so that empty cell wall cylinders remain attached to the ends of the daughter filaments, falsely suggesting the presence of a sheath. The taxonomy of Herpetosiphon is discussed in detail: The organism has been described before as Flexibacter giganteus. It is proposed to abandon the species H. aurantiacus in favor of H. giganteus, but to retain the genus Herpetosiphon. An improved definition of the genus is given.     

Factors controlling the binding of two protons per electron transferred through the ubiquinone and cytochrome b/c2 segment of Rhodopseudomonas sphaeroides chromatophores.

1. On every turnover, 2.0 protons can be bound by the membrane for each single electron moving through the Q-b/c2 oxidoreductase. 2. One proton (H+II) binding reaction is, and one (H+I) is not, sensitive to antimycin. 3. The redox states of electron transfer components other than the proton binding agents can affect both the rate of proton uptake and the apparent pK values on the agents binding the protons. 4. The presence of valinomycin under certain well-defined conditions can strongly influence the value of the measured pK on the H+II binding agent.     

An investigation into the feasibility of using yeast protoplasts to study the ion transport properties of the plasma membrane

A method for studying ion uptake in enzymatically isolated protoplasts from the yeast, Saccharomyces cerevisiae, is described. The kinetics of K⁺ and Rb⁺ uptake, metabolic proton extrusion and cell electrophoretic mobility bave been determined. Enzymic removal of the cell wall does not significantly alter the above-mentioned properties of the yeast cells. It is concluded that studies of these properties can be performed equally well with intact yeast cells or protoplasts. However, in studies aimed at determining effects of complex organic substances, e.g., antibiotics, on plasma membrane function the use of protoplasts is recommended. The effectiveness of the antibiotic, Dio-9, for example, in reversing the metabolic proton extrusion into a net proton influx is at least 50 times higher after enzymic removal of the yeast cell wall.     

Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for 'ping pong' kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione

Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pK(a) of 8.6. Only a small proportion of C14 is reactive with 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS(-) (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site.     

Myelin basic protein binds microtubules to a membrane surface and to actin filaments in vitro: effect of phosphorylation and deimination

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. It assembles actin filaments and microtubules, can bind actin filaments and SH3-domains to a membrane surface, and may be able to tether them to the oligodendrocyte membrane and participate in signal transduction in oligodendrocytes/myelin. In the present study, we have shown that the 18.5 kDa MBP isoform can also bind microtubules to lipid vesicles in vitro. Phosphorylation of MBP at Thr94 and Thr97 (bovine sequence) by MAPK, and deimination of MBP (using a pseudo-deiminated recombinant form), had little detectable effect on its ability to polymerize and bundle microtubules, in contrast to the effect of these modifications on MBP-mediated assembly of actin. However, these modifications dramatically decreased the ability of MBP to tether microtubules to lipid vesicles. MBP and its phosphorylated and pseudo-deiminated variants were also able to bind microtubules to actin filaments. These results suggest that MBP may be able to tether microtubules to the cytoplasmic surface of the oligodendrocyte membrane, and that this binding can be regulated by post-translational modifications to MBP. We further show that MBP appears to be co-localized with actin filaments and microtubules in cultured oligodendrocytes, and also at the interface between actin filaments at the leading edge of membrane processes and microtubules behind them. Thus, MBP may also cross-link microtubules to actin filaments in vivo.     

Aspects of the chemistry of d-glyceraldehyde 3-phosphate dehydrogenase

Crystalline d-glyceraldehyde 3-phosphate dehydrogenase from lobster tail contains 4 moles of NAD(+) bound and reacts specifically with 4 moles of iodoacetic acid/mole of tetramer. The essential thiol group of d-glyceraldehyde 3-phosphate dehydrogenase appears to react with iodoacetic acid with a rate constant for the overall process that is independent of the extent of carboxymethylation. The d-glyceraldehyde 3-phosphate dehydrogenase-NAD(+) absorption band has a variable molar extinction coefficient in the presence of phosphate that may be correlated with a proton dissociation of pK 6.86. The binding of NAD(+) to d-glyceraldehyde 3-phosphate dehydrogenase weakens as alkylating agents react with the enzyme, and NAD(+) promotes the reactivity of the essential thiol group. It is suggested that, on binding to d-glyceraldehyde 3-phosphate dehydrogenase, NAD(+) lowers the pK of the essential thiol group, resulting in a catalytic role of NAD(+) in the reaction catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. If this theory is correct, then it is likely that a proton will be liberated during the phosphorolysis of the acyl-enzyme rather than in the redox step.     

Involvement of an Extracellular Glucan Sheath during Degradation of Populus Wood by Phanerochaete chrysosporium

Observations by transmission electron microscopy of wood samples of Populus tremula inoculated with the white rot fungus Phanerochaete chrysosporium showed that, at certain stages of their growth cycle, hyphae were encapsulated by a sheath which seems to play an active role in the wood cell wall degradation. Chemical and immunochemical techniques and C nuclear magnetic resonance spectroscopy were applied to demonstrate the beta-1,3-1,6-d-glucan nature of the sheath. Double-staining methods revealed the interaction between the extracellular peroxidases involved in lignin degradation and the glucan mucilage. The glucan was also shown to establish a material junction between the fungus and the wood cell wall. It was concluded that, by means of these interactions, the sheath provides a transient junction between the hyphae and the wood, thus establishing a point of attachment to the site of the degradation. The association of peroxidases to the glucan matrix is in favor of the role of the sheath as a supporting structure. Furthermore, that the sheath was hydrolyzed during the attack demonstrated its active role both in providing the H(2)O(2) necessary to the action of peroxidases and in providing a mode of transport of the fungal enzymes to their substrates at the surface of the wood cell wall.     

Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.     

Binding of bacterial toxins to glycoproteins in the envelopes of rainbow trout eggs

Summary The ability of the vitelline and fertilization envelopes of rainbow trout eggs to trap toxins was investigated using cholera enterotoxin B and staphylococcal enterotoxin B in cytochemical or immunocytochemical experiments. Extracts from both envelopes were investigated by immunoblot analysis to identify toxin-binding proteins after SDS-PAGE. Binding studies of cholera enterotoxin B to vitelline envelopes and fertilization envelopes revealed a greater reactive intensity in the former. Treatment with neuraminidase enhanced the reactive intensity (or deposit) in the vitelline envelope and fertilization envelope outermost layers, with more conspicuous reactivity in the former. Cytochemical experiments showed that exogenous ganglioside GM₁ considerably enhanced cholera enterotoxin B binding to vitelline and fertilization envelopes. This enhancement was shown by an intense reactivity following the occurrence of new binding sites on the vitelline envelope inner surface and the inner wall of the zona radiata, a simultaneous extreme reduction in the reactivity of the vitelline envelope outermost layer, and a striking increase in reactive products in the fertilization envelope outermost layer. The surface region of the vitelline or fertilization envelope outermost layer was the binding site for staphylococcal enterotoxin B, and neuraminidase treatment caused a considerable reduction of reactive products in these areas. Immunoblot analysis of cholera enterotoxin Bor staphylococcal enterotoxin B-binding substances in extracts from the vitelline envelopes or fertilization envelopes demonstrated that the great majority of the binding substances are glycoproteins. The present results suggest that glycoproteins constituting the vitelline envelope or fertilization envelope may contribute to the protection of the egg itself or the embryo by trapping noxious toxins.     

Enzymic and immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site.

Previous studies from this laboratory on the immunochemistry of specific chemical derivatives of native lysozyme and of the two disulfide peptide 62-68 (Cys 64-Cys 80) 74-97 (Cys 76-Cys 94) (i.e. (SS)2-peptide), have established an antigenic reactive site to comprise the spatially contiguous surface residues: Trp 72, Lys 97, Lys 96, Asn 93, Thr 89 and Asp 87. In the present work, the identity of the site was verified by an entirely different and novel approach. The aforementioned amino acids were linked directly into a single linear peptide with an intervening spacer where appropriate and substituting phenylalanine for tryptophan (i.e. Phe-Gly-Lys-Asn-Thr-Asp). This peptide (which does not exist in native lysozyme but simulates a surface region of the protein) possessed a remarkable inhibitory activity towards the reaction of lysozyme with its antisera. The immunochemical reactivity of the peptide was equal to the maximum expected reactivity of the site (i.e. a third of the total antigenic reactivity of lysozyme). These findings define quite conclusively and accurately the reactive site which is clearly composed of spatially adjacent residues that are distant in sequence reacting as if in direct linear linkage. The unequivocal establishment of this concept indicates that antigenic sites need not always be composed of residues in direct peptide linkage in the sequence. The nature of the site may depend on the protein. This unorthodox attack at the problem provides a novel and powerful approach for final delineation of the antigenic reactive sites (and perhaps other types of binding sites) in native proteins, following the completion of accurate narrowing down by chemical methods.     

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.

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.