Iron removal from monoferric human serum transferrins by 1, 2-dimethyl-3-hydroxypyridin-4-one, 1-hydroxypyridin-2-one and acetohydroxamic acid.

The kinetics of iron removal from both forms of human serum monoferric transferrin by three ligands, 1, 2-dimethyl-3-hydroxypyridin-4-one (L1), 1-hydroxypyridin-2-one and acetohydroxamic acid, have been evaluated at pH 7.4 and 25.0 degreesC. In almost all cases the rate of iron removal follows simple saturation kinetics with respect to the ligand concentration. No spectroscopically distinct intermediates are observed during the iron removal reaction, which is consistent with a mechanism in which the rate-limiting step in iron removal is a protein conformational change. In the presence of chloride or perchlorate, most systems continue to follow simple saturation kinetics, but with significantly different kmax values. Chloride accelerates iron release from both transferrin binding sites, while perchlorate accelerates iron release from the C-terminal site but retards iron release from the N-terminal site. When the hydrochloride salt of L1 is used to prepare the L1 stock solution, the allosteric effect of the chloride produces a continuing increase in the rate of iron removal with increasing ligand concentration, so that one no longer observes simple saturation kinetics. A least squares fit of kobs vs. the ligand concentration for L1.HCl shows that the allosteric effect of the chloride not only enhances the first-order term for iron removal but also doubles the apparent kmax for the saturation term. This supports the view that allosteric binding of anionic ligands contributes to the observed variation in kmax among different ligands. A detailed description of this allosteric effect is not yet possible because the effect varies significantly from system to system, depending upon the specific anion that is binding at the allosteric site, the ligand that is used to remove the iron, and the transferrin lobe from which iron is removed.

Binding of monovalent anions to human serum transferrin.

Serum transferrin is the protein whose primary function is to bind iron and transport it through the blood. Apotransferrin has two specific metal-binding sites that bind a variety of metal ions in addition to the ferric ion. The distinguishing feature of the transferrins is that a "synergistic" bicarbonate anion is bound along with the metal ion to form a stable Fe(3+)-CO3-Tf ternary complex. Previous research has shown that apotransferrin will also bind divalent anions such as phosphate and sulfate. Difference UV spectroscopy has now been used to show that a series of monovalent anions bind weakly to apotransferrin. Equilibrium constants for the binding of chloride, perchlorate, bromide, fluoride and Hepes have been calculated. A reaction scheme for the binding of anions is proposed which predicts that the binding of the nonsynergistic anions to apotransferrin will interfere with metal binding by competing directly with the binding of the synergistic bicarbonate anion. Difference UV data are presented which demonstrate this type of competition between nonsynergistic anions and Tb3+. Competition from the nonsynergistic anions follows the order HPO4(2-) > SO4(2-) approximately F- > ClO4- approximately Cl- approximately Br-. Speciation calculations have been performed to determine the concentrations of anion-apotransferrin complexes in Hepes and Tris buffers and in human serum and to estimate the extent to which competition from anions in the buffer will interfere with metal-binding to apotransferrin.

Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules.

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.

Comparison of whole blood serotonin and platelet MAO in children with schizophrenia and major depressive disorder.

Whole blood serotonin and platelet monoamine oxidase (MAO) activity in boys with schizophrenia, schizotypal personality disorder, or major depressive disorder was compared with that of boys serving as controls. Boys with schizophrenia and schizotypal personality disorder had significantly higher platelet MAO than boys with major depressive disorder or controls. Boys with major depressive disorder had lower whole blood serotonin than boys with schizophrenia or schizotypal personality disorder.

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin.

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.

Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids.

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].

Electron paramagnetic resonance and difference ultraviolet studies of Mn2+ binding to serum transferrin.

Serum transferrin is the mammalian protein whose normal function is to transport ferric ions through the blood among sites of absorption, storage, and utilization. It has two specific metal-binding sites that bind a variety of metal ions in addition to ferric ion. The macroscopic equilibrium constant for the binding of the first equivalent of Mn2+ to apotransferrin has been determined by electron paramagnetic resonance spectroscopy (EPR) to be logKM1 = 4.06 +/- 0.13 at pH 7.4 in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (Hepes). An equilibrium constant for nonspecific binding of Mn2+ to apotransferrin of logKns = 2.93 +/- 0.13 has also been obtained by using EPR. Binding of Mn2+ to apotransferrin and to both C- and N-terminal nonferric transferrin has also been studied by difference UV spectroscopy. The second stepwise macroscopic equilibrium constant for the formation of Mn2Tf is logKM2 = 2.96 +/- 0.13. The site-specific microconstants for Mn2+ binding are logkN = 3.13 +/- 0.09 for the N-terminal site and logkC = 3.80 +/- 0.09 for the C-terminal site. There does not appear to be any significant cooperativity between the two sites with respect to metal binding. An equilibrium model for the speciation of Mn2+ in serum has been developed which estimates that almost 90% of Mn2+ is bound to serum proteins, but only approximately 1% is bound to transferrin. The weak binding of Mn2+ to apotransferrin and the obvious inability of transferrin to compete with albumin indicates that the appearance of Mn-transferrin as a major serum species in vivo must involve oxidation of the metal to form the much more stable Mn(3+)-transferrin complex. The computer model confirms that albumin has a sufficient binding affinity to complex most of the Mn(II) in serum in competition with the common low molecular weight ligands in serum. However, there is insufficient data to rule out the possibility that some other protein, such as alpha 2-macroglobulin, may compete with albumin for Mn(II).

The bicarbonate-dependence of zinc(II)-transferrin binding.

The binding of zinc(II) to human serum transferrin has been studied as a function of the solution concentration of sodium bicarbonate in 100 mM, pH 7.4 hepes buffer at 25 degrees C. The apparent molar absorptivity of the zinc-transferrin complex has been determined from the initial slopes of titration curves of delta epsilon versus the ratio of [Zn]/[Tf]. This absorptivity represents the difference between the positive absorbance of the ternary Zn-HCO3-Tf species in the sample cuvette and the negative absorbance of binary HCO3-Tf species in the reference cuvette. Higher concentrations of bicarbonate increase the degree of saturation of apo-Tf with bicarbonate and thus increase the apparent absorptivity of the zinc-Tf complex. Titrations of apo- and monoferric transferrins with bicarbonate indicate that there is little, if any, difference in the bicarbonate binding constants of the two specific transferrin binding sites. An equilibrium constant of log K = 2.49 has been used to calculate the degree of saturation of the C-terminal binding site with bicarbonate. The zinc-binding affinity of this site depends linearly on this degree of saturation. The scatter in the zinc-binding constants of the weaker N-terminal site precludes a similar analysis of the bicarbonate-dependence of binding at this site. The results strongly support the previous proposal that binding of the synergistic bicarbonate anion is responsible for the uv absorption observed upon addition of bicarbonate to apoTf.

Binding of vanadate to human serum transferrin.

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.

Behavior of vanadate and vanadyl ion in canine blood.

Radiolabeled vanadium as either vanadyl ion or vanadate ion was injected intravenously into adult beagle dogs, and blood samples were collected at various times up to 48 hr post injection. For each sample, the distribution of vanadium between the cells and the plasma was determined, and the plasma was analyzed by electrophoresis to identify specific vanadium-binding proteins. Initially, vanadyl ion left the bloodstream more rapidly than vanadate, but the rates equalized after about 5 hr. A significant fraction of the vanadium in blood was associated with the cellular component following injection of both forms of vanadium. About 77% of the plasma vanadium was eventually bound by the serum iron transport protein transferrin, regardless of the vanadium species initially injected. For both vanadyl and vanadate, about 30 hr were required to reach the maximum degree of transferrin binding.

Steric restrictions on the binding of large metal ions to serum transferrin.

Apotransferrin in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid at 25 degrees C and pH 7.4 was titrated with acidic solutions of Lu3+, Tb3+, and Eu3+. Metal binding at the two specific metal-binding sites of transferrin was followed from changes in the difference UV spectra at 245 nm. The binding of Tb3+ was also followed from changes in the fluorescence emission spectrum at 549 nm. Apotransferrin was titrated with solutions containing varying ratios of the metal ion and the competitive chelating agent nitrilotriacetic acid, and metal-transferrin binding constants were calculated by nonlinear least-squares fits of the absorbance as a function of titrant added. The sequential carbonate-independent equilibrium constants for the binding of two metal ions are log KM1 = 11.08 and log KM2 = 7.93 for Lu3+, log KM1 = 11.20 and log KM2 = 7.61 for Tb3+, and log KM1 = 9.66 and log KM2 = 7.27 for Eu3+. Titrations of both C-terminal and N-terminal monoferric transferrins indicate that all of these metal ions bind more strongly to the C-terminal binding site. The trend in log K values as a function of the lanthanide ionic radius has been evaluated both by plots of log K versus the metal ion charge/radius ratio and by linear free-energy relationships in which binding constants for complexes of the larger lanthanides are plotted versus the binding constants for complexes with the smallest lanthanide, Lu3+. Both methods indicate that there is a sharp drop in the binding constants for the C-terminal binding site for metals larger than Tb3+. This decrease is attributed to a steric hindrance to the binding of the larger cations. The steric effect is not as strong for metal binding at the N-terminal site. As a result, the selectivity for binding to the C-terminal site, which is quite high for the smaller lanthanides, drops sharply on going from Tb3+ to Nd3+.

Site selectivity in the binding of inorganic anions to serum transferrin.

Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding.

Traumatic winging of the scapula.

Fourteen patients with traumatic winging of the scapula were reviewed, all of whom had had injuries producing sudden depression of the shoulder girdle from either a direct blow to the top of the shoulder or downward traction on the arm. The diagnosis was commonly missed for a considerable interval. Seven patients recovered spontaneously within six months of injury. Three of the other seven patients were treated by reattachment of the insertion of the sternal portion of the pectoralis major muscle via a fascia lata graft to the lower pole of the scapula. In one of these patients a reoperation was needed, but all three ultimately recovered satisfactory function of the shoulder. Anatomical studies suggested that the injury results from compression of the long thoracic nerve against the second rib and not from entrapment of the nerve by the scalenus medius muscle.

Differences in heart rate and blood pressure in children with conduct disorder, major depression, and separation anxiety.

Heart rate and blood pressure of children and adolescents admitted to a psychiatric hospital were compared among those diagnosed conduct disorder, major depressive disorder, and separation anxiety disorder. Subjects with conduct disorder had a lower heart rate compared to subjects without a conduct disorder diagnosis; and subjects with separation anxiety disorder had higher heart rate and systolic blood pressure compared to subjects without an anxiety disorder diagnosis. Subjects with major depressive disorder had higher systolic blood pressure than subjects with conduct disorder but no difference in heart rate. The findings are consistent with conduct disorder being associated with decreased noradrenergic function and anxiety/depressive disorder being associated with increased noradrenergic function.

Attention deficit disorder symptoms and urine catecholamines.

The symptoms of hyperactivity, impulsivity, and concentration deficits associated with attention deficit disorder (ADD) may be related, in part, to alterations in dopaminergic and noradrenergic functioning. In this study we correlate the above symptoms with 24-hour urinary catecholamines and their metabolites in emotionally disturbed boys divided into two groups based on their plasma dopamine-beta-hydroxylase (DBH) activities and also divided into the following diagnostic groups: conduct disorder, undersocialized; conduct disorder, socialized; and subjects without conduct disorder. Boys in the low DBH group showed significant correlations between the ADD symptoms and the biochemical measures. The low DBH group may be more genetically homogeneous with regard to catecholamine function, making relationships between catecholamine function and behavior more visible. The group of boys with conduct disorder, socialized had higher 24-hour urinary norepinephrine and vanillylmandelic acid output. The relationship between monoamines and their metabolites appeared to differ among diagnostic groups.

Factors affecting the prognosis of brachial plexus injuries.

The clinical results in a series of 131 patients with 134 brachial plexus injuries were analysed to determine the factors affecting prognosis. Isolated injuries to the upper trunk had the best prognosis, but the prognoses of isolated injuries to the cords, upper roots and lower trunk were not as good. Complete injuries of the plexus had the worst prognosis. Pain which persisted for more than six months was a bad prognostic sign for neurological recovery regardless of the location of the lesion. Horner's syndrome was not always accompanied by a bad prognosis. Operation did not affect the prognosis except in open lacerations. A pseudomeningocele detected by myelography usually precluded recovery in the root at the level of the pseudomeningocele.

Recurrent giant-cell tumour after en bloc excision of the distal radius and fibular autograft replacement.

We report two patients, each with a giant-cell tumour of the distal radius treated by curettage and bone grafting. Local recurrence of the tumour occurred in the autograft and in the adjacent soft tissues in both patients, and was successfully treated by local excision; one patient also had radiation therapy. Both remain well 20 years and five years later.

Treatment of winged scapula by pectoralis major transfer.

We report the transfer of the sternal part of the pectoralis major to the lower pole of the scapula in 15 patients with winged scapula. At follow-up after 1 to 16 years nine had a satisfactory result and were gainfully employed, though in four of these re-operation had been necessary. Two patients had fair results; the transplant functioned, but they had limited voluntary control. Four were failures: two had had paralysis of other shoulder girdle muscles in addition to the serratus anterior. The indications for the operation are discussed.