Nano gel filtration reveals how fish hemoglobins release oxygen: The Root Effect.

The Root Effect is to many species of fish what the Bohr Effect is to humans regarding the release of O2 from their hemoglobins at low pH. However, Root Effect hemoglobins accomplish this more extensively than human adult hemoglobin in order to satisfy the diverse oxygen requirements in fish. To understand this difference between fish and human hemoglobins, we studied their subunit interface strengths using very low (nanomolar) concentrations, referred to as nano gel filtration. Root Effect hemoglobins in their CO form dissociate in a tetramer-monomer equilibrium. In contrast, tetramers and dimers but no monomers are found for adult human hemoglobin consistent with its well known tetramer-dimer equilibrium. By analogy to the human variant Hb Kansas and a similar recombinant Hb, both of which readily release oxygen due to an unstable oxygenated structure, the mechanism proposed is that oxygenated Root Effect tetramers release their oxygen to form energetically stable deoxygenated tetramers rather than dissociate to energetically unfavorable oxygenated dimers with labile interfaces. In contrast, the strong binding of CO permits observation of dissociation to monomers, thus revealing an intrinsic property of Root Effect fish hemoglobins enabling it to function as an oxygen pump.

Embryonic and Fetal Human Hemoglobins: Structures, Oxygen Binding, and Physiological Roles.

During the past two decades, significant advances have been made in our understanding of the human fetal and embryonic hemoglobins made possible by the availability of pure, highly characterized materials and novel methods, e.g., nano gel filtration, to study their properties and to correct some misconceptions. For example, whereas the structures of the human adult, fetal, and embryonic hemoglobins are very similar, it has generally been assumed that functional differences between them are due to primary sequence effects. However, more recent studies indicate that the strengths of the interactions between their subunits are very different leading to changes in their oxygen binding properties compared to adult hemoglobin. Fetal hemoglobin in the oxy conformation is a much stronger tetramer than adult hemoglobin and dissociates to dimers 70-times less than adult hemoglobin. This property may form the basis for its protective effect against malaria. A major source of the increased strength of fetal hemoglobin resides within the A-helix of its gamma subunit as demonstrated in studies with the hybrid hemoglobin Felix and related hybrids. Re-activating fetal hemoglobin synthesis in vivo is currently a major focus of clinical efforts designed to treat sickle cell anemia since it inhibits the aggregation of sickle hemoglobin. The mechanisms for both the increased oxygen affinity of fetal hemoglobin and its decreased response to DPG have been clarified. Acetylated fetal hemoglobin, which makes up 10-20% of total fetal hemoglobin, has a significantly weakened tetramer structure suggesting a similar role for other kinds of protein acetylation. Embryonic hemoglobins have the weakest tetramer and dimer structures. In general, the progressively increasing strength of the subunit interfaces of the hemoglobin family during development from the embryonic to the fetal and ultimately to the adult types correlates with their temporal appearance and disappearance in vivo, i.e., ontogeny.

Phosphorylation of Serine Induces Lysine p Ka Increases in Histone N-Termini and Signaling for Acetylation. Transcription Implications.

The mild acetylating agent, methyl acetyl phosphate, is used to estimate the p Ka values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys ε amines have p Ka values in the range of 7.8-8.3, which are much lower than the currently assumed values. When Ser-1 is phosphorylated, the p Ka values of these Lys amines are elevated to the range of 8.8-10.3, thus providing the rationale for reports that they are then better substrates for acetyltransferases. Thus, reversal of suppressed p Ka values of Lys ε amines by Ser phosphorylation represents the basis for signaling in histone N-terminal tails to promote hyperacetylation, which is a hallmark of transcriptionally active euchromatin. In contrast, a state of hypoacetylation is present in the absence of phosphorylation as in transcriptionally inactive heterochromatin. A novel approach for estimating p Ka values based on a linkage between the Henderson-Hasselbalch and Michaelis-Menten equations indicates that the p Ka values of the Lys ε amines in H3 and H4 N-terminal tails have a highly variable charge gradient dependent on the location and proximity to the phosphorylation site.

Characterization of a novel cysteine-rich antifungal protein from Fusarium graminearum with activity against maize fungal pathogens.

Filamentous fungi are an invaluable source for biocontrol strategies and for production and development of different antifungal polypeptides. Within this context, cysteine-rich antifungal AFP-like peptides stand out among many different antimicrobial compounds given their production easiness, stability, versatility, and efficacy. AFP from Aspergillus giganteus represents the hallmark of this still increasing family of antifungal polypeptides. Close in silico analyses of the Fusarium graminearum genome revealed the presence of an AFP-like peptide, here designated as FgAFP. This new peptide was cloned, produced in the yeast Pichia pastoris, and characterized. The results obtained showed its strong and specific antifungal activity against several well-recognized maize pathogens, but inefficacy against F. oxysporum, which has not been described as a natural biological competitor of other fungal pathogens assayed. All results together suggest that this small peptide is an important factor for the fungal interplays involved in maize infection and reveals unforeseen potential biotechnological applications for FgAFP in maize production and storage.

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities.

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14, and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pKa of these Lys residues induced by phosphorylation; basicities increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14, and Lys-18 have lowered pKa values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate-Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding.

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect.

Intrinsic regulation of hemoglobin expression by variable subunit interface strengths.

The expression of the six types of human Hb subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the 'extrinsic' component of regulation). Here, we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the 'intrinsic' component of regulation). The adult Hb dimers have the strongest subunit interfaces and the embryonic Hbs the weakest, with fetal Hbs being of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these Hb types form functional O(2) -binding tetramers consistent with gene switching.

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces.

Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low-Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties.

Chiral discrimination among aminotransferases: inactivation by 4-amino-4,5-dihydrothiophenecarboxylic acid.

Mechanism-based inhibitors such as cycloserine and gabaculine can inactivate aminotransferases via reactions of the compounds with the pyridoxal phosphate cofactor forming an irreversible adduct. The reaction is chirally specific in that any one enzyme usually only recognizes one enantiomer of the inactivator. For instance, l-aspartate aminotransferase (l-AspAT) is inactivated by 4-amino-4,5-dihydro-2-thiophenecarboxylic acid (ADTA), however, only by the S-isomer. We have now shown that d-amino acid aminotransferase (d-a-AT) is irreversibly inactivated by the R-isomer of the same compound. The X-ray crystal structure (PDB code: 3LQS ) of the inactivated enzyme shows that in the product the enzyme no longer makes a Schiff base linkage to the pyridoxal 5'-phosphate (PLP) cofactor, and instead the compound has formed a derivative of the cofactor. The adduct is similar to that formed between d-cycloserine and d-a-AT or alanine racemase (Ala-Rac) in that the thiophene ring of R-ADTA is intact and seems to be aromatic. The plane of the ring is rotated by nearly 90 degrees with respect to the plane of the pyridine ring of the cofactor, in comparison with the enzyme inactivated by cycloserine. Based on the structure of the product, the mechanism of inactivation most probably involves a transamination followed by aromatization to form an aromatic thiophene ring.

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris.

The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (lf-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP *) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP * produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches.

Energetic differences at the subunit interfaces of normal human hemoglobins correlate with their developmental profile.

A previously unrecognized function of normal human hemoglobins occurring during protein assembly is described, i.e. self-regulation of subunit pairings and their durations arising from the variable strengths of their subunit interactions. Although many mutant human hemoglobins are known to have altered subunit interface strengths, those of the normal embryonic, fetal, and adult human hemoglobins have not been considered to differ significantly. However, in a comprehensive study of both types of subunit interfaces of seven of the eight normal oxy human hemoglobins, we found that the strengths, i.e., the free energies of the tetramer-dimer interfaces, contrary to previous reports, differ by 3 orders of magnitude and display an undulating profile similar to the transitions ("switches") of various globin subunit types over time. The dimer interface strengths are also variable and correlate linearly with their developmental profile. Embryonic hemoglobins are the weakest; fetal hemoglobin is of intermediate strength, and adult hemoglobins are the strongest. The pattern also correlates generally with their different O(2) affinities and responses to allosteric regulatory molecules. Acetylation of fetal hemoglobin weakens its unusually strong subunit interactions and occurs progressively as its level of expression diminishes and adult hemoglobin A formation begins; a causal relationship is suggested. The relative contributions of globin gene order and competition among subunits due to differences in their interface strengths were found to be complementary and establish a connection among genetics, thermodynamics, and development.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell.

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (zeta(2)beta(2)) < Hb Portland-1 (zeta(2)gamma(2)) approximately equal Hb Gower-1 (zeta(2)epsilon(2)) < Hb Gower-2 (alpha(2)epsilon(2)) < HbF(1) < HbF (alpha(2)gamma(2)) < HbA(2) (alpha(2)delta(2)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic --> fetal --> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity.

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the alpha- or the beta-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the alpha-subunits of liganded hemoglobin results in a significantly increased basicity (increased pK(a) values) and a reduction in the strength of subunit interactions at the allosteric tetramer-dimer interface. Cooperativity in O(2) binding is also decreased. Substitution of Ala at the N-terminals of the beta-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer-dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the alpha-subunit, the slope of the plot of the tetramer-dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the alpha-subunits is less extensive than that of the beta-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

Hemoglobin Jamaica plain--a sickling hemoglobin with reduced oxygen affinity.

A baby girl presented with symptomatic sickle cell disease exacerbated by mild hypoxemia, despite a newborn-screening diagnosis of sickle cell trait. DNA sequencing of the beta globin gene revealed that her maternal beta globin allele was normal. Her paternal allele had not only the expected sickle-trait mutation, betaGlu6Val, but also a second, charge-neutral mutation, betaLeu68Phe. Analysis of the patient's hemoglobin revealed that the double-mutant protein, which we called "hemoglobin Jamaica Plain," had severely reduced oxygen affinity. Structural modeling suggested destabilization of the oxy conformation as a molecular mechanism for sickling in a heterozygote at an ambient partial pressure of oxygen.

Human erythrocyte membrane band 3 protein influences hemoglobin cooperativity. Possible effect on oxygen transport.

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.

Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids.

As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer.

N-terminal contributions of the gamma-subunit of fetal hemoglobin to its tetramer strength: remote effects at subunit contacts.

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.

Predicting Performance on the National Athletic Trainers' Association Board of Certification Examination From Grade Point Average and Number of Clinical Hours.

OBJECTIVE: To determine whether grade point average, hours of clinical education, or both are significant predictors of performance on the National Athletic Trainers' Association Board of Certification examination and whether curriculum and internship candidates' scores on the certification examination can be differentially predicted. DESIGN AND SETTING: Data collection forms and consent forms were mailed to the subjects to collect data for predictor variables. Subject scores on the certification examination were obtained from Columbia Assessment Services. SUBJECTS: A total of 270 first-time candidates for the April and June 1998 certification examinations. MEASUREMENTS: Grade point average, number of clinical hours completed, sex, route to certification eligibility (curriculum or internship), scores on each section of the certification examination, and pass/fail criteria for each section. RESULTS: We found no significant difference between the scores of men and women on any section of the examination. Scores for curriculum and internship candidates differed significantly on the written and practical sections of the examination but not on the simulation section. Grade point average was a significant predictor of scores on each section of the examination and the examination as a whole. Clinical hours completed did not add a significant increment for any section but did add a significant increment for the examination overall. Although no significant difference was noted between curriculum and internship candidates in predicting scores on sections of the examination, a significant difference by route was found in predicting whether candidates would pass the examination as a whole (P =.047). Proportion of variance accounted for was less than R(2) = 0.0723 for any section of the examination and R(2) = 0.057 for the examination as a whole. CONCLUSIONS: Potential predictors of performance on the certification examination can be useful to athletic training educators in assisting students. These findings must be used cautiously because of the low proportion of explained variance. Low R(2) values suggest that the largest contributors to performance on the examination were not identified in this study. Although the results of this study support the decision to discontinue the internship route to certification, continued research focusing on identification and investigation of the constructs that contribute to examination success is needed.