Biomedical subjects

M Bolognesi

Publications and source records attributed to M Bolognesi.

At least 37 records · Page 2Linked to original sources

PubMed2001

Noninvasive grading of the severity of portal hypertension in cirrhotic patients by echo-color-Doppler.

The possibility of estimating portal hypertension combining splenic Doppler pulsatility index (PI), which has been shown strictly to be related to portal resistance, and portal blood flow (PBF) was evaluated. Hepatic venous pressure gradient, and splanchnic Doppler parameters were measured in 40 cirrhotic patients. A formula to assess the severity of portal hypertension from Doppler parameters was calculated in a training group of 19 patients, and then this formula was tested to prospectively predict the degree of portal pressure in 21 further patients. In the training group, the regression of portal resistance over splenic PI was calculated and individual values of estimated portal resistance were obtained for every patient of the test group. From them and from observed values of PBF, an estimated value of portal pressure was calculated. The following formula was obtained [(0.066*splenic PI -0.044)*PBF]. In the second group, predicted and observed portal pressure were well correlated (r = 0.71, p = .0003). A good accuracy for the prediction of mild or severe portal hypertension was obtained.

Adult↗

PubMed2001

Escherichia coli GlpE is a prototype sulfurtransferase for the single-domain rhodanese homology superfamily.

BACKGROUND: Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. RESULTS: Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. CONCLUSIONS: Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.

Amino Acid Sequence↗

PubMed2001

Structural determinants of CCR5 recognition and HIV-1 blockade in RANTES.

Certain chemokines act as natural antagonists of human immunodeficiency virus (HIV) by blocking key viral coreceptors, such as CCR5 and CXCR4, on the surface of susceptible cells. Elucidating the structural determinants of the receptor-binding and HIV-inhibitory functions of these chemokines is essential for the rational design of derivative molecules of therapeutic value. Here, we identify the structural determinants of CCR5 recognition and antiviral activity of the CC chemokine RANTES, showing that critical residues form a solvent-exposed hydrophobic patch on the surface of the molecule. Moreover, we demonstrate that the biological function is critically dependent on dimerization, resulting in the exposure of a large ( approximately 180 A2), continuous hydrophobic surface. Relevant to the development of novel therapeutic approaches, we designed a retroinverted RANTES peptide mimetic that maintained both HIV- and chemotaxis-antagonistic functions.

Alanine↗

PubMed2001

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.

Animals↗

PubMed2001

Crystallization and preliminary crystallographic studies on the large extracellular domain of human CD81, a tetraspanin receptor for hepatitis C virus.

The large extracellular domain of CD81, a member of the tetraspanin family and a receptor protein for hepatitis C virus envelope E2 glycoprotein, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.6 A resolution were obtained at the ID14 beamline of the European Synchrotron Radiation Facility from a flash-frozen crystal at 100 K. The crystals belong to space group P2(1), with unit-cell parameters a = 31.5, b = 77.2, c = 38.5 A, beta = 107.4 degrees, and are likely to contain two extracellular domains (2 x 99 residues) per asymmetric unit.

Antigens, CD↗

PubMed2001

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii Rhodanese.

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.

Azotobacter vinelandii↗

PubMed2001

Inhibition of cysteine protease activity by NO-donors.

Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed.

Animals↗

PubMed2000

Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.

GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.

Amino Acid Substitution↗

PubMed2000

Single mutation at the intersubunit interface confers extra efficiency to Cu,Zn superoxide dismutase.

The Val28-->Gly single mutant at the subunit interface of Cu,Zn superoxide dismutase from Photobacterium leiognathi displays a k(cat)/K(M) value of 1.7x10(10) M(-1) s(-1), twice that of the native enzyme. Analysis of the three-dimensional structure indicates that the active site Cu,Zn center is not perturbed, slight structural deviations being only localized in proximity of the mutation site. The enzyme-substrate association rate, calculated by Brownian dynamics simulation, is identical for both enzymes, indicating that the higher catalytic efficiency of the Val28-->Gly mutant is not due to a more favorable electrostatic potential distribution. This result demonstrates the occurrence of an intramolecular communication between the mutation site and the catalytic center, about 18 A away and indicates a new strategy to encode extra efficiency within other members of this enzymatic family.

Amino Acid Substitution↗

PubMed2000

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene.

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.

Amino Acid Sequence↗

PubMed2000

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.

Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a 'two-over-two' alpha-helical sandwich, reflecting an unprecedented editing of the classical 'three-over-three' alpha-helical globin fold. Based on specific Gly-Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

Amino Acid Motifs↗

PubMed2000

The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights the evolutionary relationship between the rhodanese and phosphatase enzyme families.

Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a rhodanese. The crystal structure of the A. vinelandii rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.

Amino Acid Sequence↗

PubMed2000

Enhancement of the HIV-1 inhibitory activity of RANTES by modification of the N-terminal region: dissociation from CCR5 activation.

Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infection, their inherent proinflammatory activity may limit a therapeutic use. To elucidate whether the antiviral and signaling functions of RANTES can be dissociated, several recombinant analogues mutated at the N terminus were generated and functionally compared with the wild-type (WT) molecule, as well as with three previously described mutants. Substitution of selected residues within the N-terminal region caused a marked loss of antiviral potency. By contrast, two unique analogues (C1.C5-RANTES and L-RANTES) exhibited an increased antiviral activity against different CXCR4-negative HIV-1 isolates grown in primary mononuclear cells or in macrophages. This enhanced HIV-blocking activity was associated with an increased binding affinity for CCR5. Both C1.C5-RANTES and L-RANTES showed a dramatically reduced ability to trigger intracellular calcium mobilization via CCR3 or CCR5, while potently antagonizing the action of the WT chemokine. By contrast, two previously described analogues (RANTES(3-68) and AOP-RANTES) maintained a WT ability to trigger CCR5-mediated signaling, while a third one (RANTES(9-68)) showed a dramatic loss of antiviral activity. These data demonstrate that the antiviral and signaling functions of RANTES can be uncoupled, opening new perspectives for the development of chemokine-based therapeutic approaches for HIV infection.

Anti-HIV Agents↗

PubMed2000

Long-term results of a clinical trial of nadolol with or without isosorbide mononitrate for primary prophylaxis of variceal bleeding in cirrhosis.

It is clearly established that beta-blockers decrease the risk of a first variceal bleeding in cirrhosis. We have recently shown that the addition of isosorbide mononitrate to nadolol decreases the rate of variceal bleeding in patients with cirrhosis and varices, compared with nadolol alone, after a median follow-up of 30 months. It is not established if the long-term treatment with the combination continues to be beneficial. Therefore, we assessed the long-term effect of this combination on first variceal bleeding, complications, and death. One hundred forty-six cirrhotic patients with esophageal varices included in a previously published multicenter, randomized study comparing nadolol (40-160 mg/d) with the combination nadolol plus isosorbide mononitrate (10-20 mg 3 times per day) were followed up for up to 7 years (median follow-up, 55 months). The primary end-point was variceal bleeding of any severity. Twenty-four patients (16 in the nadolol group, and 8 in the combination group) experienced variceal bleeding (log rank test, P =.02). Cumulative risk of bleeding was 29% and 12%, respectively (95% CI for the difference, 1%-23%). Two and 4 patients, respectively, had bleeding from portal hypertensive gastropathy (log rank test, P =.20). Thirty and 25 patients, respectively, died during follow-up (log rank test, P =.13). Twelve and 10 patients, respectively, had de novo occurrence of ascites during follow-up (log rank test, P =.29). In conclusion, nadolol plus isosorbide mononitrate is significantly more effective than nadolol alone in the long-term use. Side effects are few, and no deleterious effects on ascites occurrence or on survival occur after long-term use of this combination.

Adolescent↗

PubMed2000

Serine proteinase inhibition by the active site titrant N alpha-(N, N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester. A comparative study.

Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).

Animals↗

PubMed2000

The hemodynamic response to medical treatment of portal hypertension as a predictor of clinical effectiveness in the primary prophylaxis of variceal bleeding in cirrhosis.

In the prevention of variceal rebleeding, it is already established that hemodynamic response to drug treatment (decrease in hepatic venous pressure gradient [HVPG] to 12 mm Hg or by >20%) is predictive of clinical effectiveness. In primary prophylaxis very few clinical data are available. We assessed the role of the hemodynamic response to beta-blockers or beta-blockers plus nitrates in predicting clinical efficacy of prophylaxis. A total of 49 cirrhotic patients with varices at risk of bleeding, without prior variceal bleeding, were investigated by hepatic vein catheterization before and after 1 to 3 months of chronic treatment with nadolol or nadolol plus isosorbide mononitrate, and were followed during treatment for up to 5 years. A total of 30 patients (61%) were good hemodynamic responders, and among them in 12 (24%) HVPG was </=12 mm Hg during treatment. During treatment 9 patients had variceal bleeding: 7 were poor responders and 2 were good responders. The probability of bleeding at 3 years of follow-up was significantly higher in poor responders (41%) than in good responders (7%; P =.0008). No patient reaching an HVPG of 12 mm Hg or less during treatment had variceal bleeding during follow-up. Cox's regression analysis showed that poor hemodynamic response was the main factor predicting bleeding (beta = 1.91; SE(beta) = 0.80; P =.01). During follow-up 11 patients died of hepatic causes. Survival was related to Child-Pugh class and to initial value of HVPG, according to Cox's analysis. In conclusion, the assessment of hemodynamic response to drugs in terms of HVPG is the best predictor of efficacy of prophylaxis of variceal bleeding in patients treated with beta-blockers or beta-blockers plus nitrates.

Adrenergic beta-Antagonists↗

PubMed2000

Crystals of GlpE, a 12 kDa sulfurtransferase from escherichia coli, display 1.06 A resolution diffraction: a preliminary report.

The Escherichia coli sn-glycerol 3-phosphate regulon contains the glpE gene coding for a 12 kDa protein which displays a sequence and a thiosulfate:cyanide sulfurtransferase activity similar to those of rhodanese enzymes. The GlpE protein was overexpressed, purified to homogeneity and crystallized in the trigonal space group P3(1) (or P3(2)). The unit-cell parameters are a = b = 53.87, c = 30.52 A, gamma = 120 degrees. Evaluation of the crystal packing parameter establishes the presence of one molecule per asymmetric unit, with a solvent content of 42%. The GlpE crystals display very high resolution diffraction; a 1.06 A data set was collected using synchrotron radiation (lambda = 0.9102 A) with an overall completeness of 99.6%.

Crystallography, X-Ray↗

PubMed2000

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.

Crystallization↗