Binding of heparin to human antithrombin III as studied by measurements of tryptophan fluorescence.

Corrected fluorescence excitation and emission spectra of human antithrombin III have been determined. The fluorescence observed originates almost entirely from tryptophan residues. Reduction of the disulfide bonds followed by carboxymethylation did not change the fluorometric properties of the protein. The binding of heparin to antithrombin III caused a marked fluorescence enhancement by about 30% of the intrinsic protein emission intensity. Various samples of heparin yielded different binding curves. Heparin fractionated by gel filtration seemed to be bound to two sites on antithrombin III with association constants of 0.6-10(6)m-1 and 0.2-10(6)M-1 respectively. Heparin, prepared by affinity chromatography on matrix-bound antithrombin III appeared to be bound to only one site with an association constant of 2.3-10(6)M-1. Under similar conditions heparin caused no increase of the intrinsic protein emission intensity when added to reduced and carboxymethylated antithrombin III. The implications of these findings are discussed.     

A reaction cell for simultaneous measurements of fluorescence and absorption in a hemoglobin solution at various oxygen pressures

An apparatus was constructed to carry out measurements of fluorescence, optical absorption and oxygen partial pressure in a hemoglobin or other solution simultaneously, and its performance was examined. This apparatus has a rhombiform optical cell in place of the usual square optical cell used in commercially available spectrofluorometers. Fluorescence emitted at the region near the cell surface in the solution could be detected satisfactorily and easily even if the solution had strong light absorption bands at both the excitation and the emission wavelengths in the presence of high concentrations of a chromophore. This apparatus was particularly effective for studies on the interactions of a fluorescent allosteric effector with hemoglobin at various degrees of deoxygenation. Consequently, it was proved experimentally that the fluorescence of β-naphthyl triphosphate bound to hemoglobin is completely quenched. Moreover, simultaneous and continuous measurements of the oxygen-binding equilibrium of hemoglobin and the allosteric effector-binding to hemoglobin as a function of oxygen partial pressure could be satisfactorily carried out, and it is confirmed that β-naphthyl triphosphate binds not only to deoxyhemoglobin but also to fully oxygenated hemoglobin and lowers strongly the oxygen affinity of hemoglobin as an allosteric effector.     

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.     

Glycosylated minor components of human adult hemoglobin. Purification, identification, and partial structural analysis

Human hemolysate contains several minor components designated Hb A1a, Hb A1b, Hb A1c, which are post-translational modifications of the major hemoglobin component A0. Individuals with diabetes mellitus have elevated levels of Hb A1c, a hemoglobin modified with a glucose moiety at the NH2 terminus of each beta chain. A new chromatographic technique using Bio-Rex 70 is described which not only allows complete separation of Hb A1a from Hb A1b but also resolution of Hb A1a into two components, designated Hb A1a1 and Hb A1a2. Carbohydrate determinations with the thiobarbituric acid procedure revealed that Hb A1a1, Hb A1a2, and Hb A1b as well as Hb A1c were glycosylated. Total phosphate analysis revealed 2.06 and 1.01 mol of phosphorus/alphabeta dimer for Hb A1a1 and Hb A1a2 respectively; Hb A1b and Hb A1c contained no detectable phosphate. Hemoglobin incubated with D-[14C]glucose-6-P co-chromatographs precisely with Hb A1a2, strongly suggesting that Hb A1a2 is glucose-6-P hemoglobin. Levels of Hb A1a1 and Hb A1a2 are normal in individuals with diabetes mellitus. Furthermore, diabetic red cells contain normal levels of glucose-6-P. Therefore, glucose-6-P hemoglobin does not serve as a significant precursor to Hb A1c. Instead Hb A1c is formed by the direct reaction of hemoglobin with glucose. This suggests that hemoglobin can serve as a model system for nonenzymatic glycosylation of protein.     

Chlorpheniramine binding to human serum albumin by fluorescence quenching measurements.

The binding of chlorpheniramine to human serum albumin has been studied by fluorescence quenching, as a function of temperature; the experimental data could only be fitted to the Stern-Volmer modified equation. A statistical analysis of the results was performed in order to determine the significance of the constants calculated by this equation, as well as their thermodynamic parameters. The chlorpheniramine binding to human serum albumin accounts for almost half of the binding of this antihistaminic agent to human plasma proteins.     

Energy metabolism in relation to oxygen partial pressure in human skeletal muscle during exercise.

1. The intramuscular oxygen partial pressure (pO2) in human gastrocnemius muscle was monitored during exercise and compared with metabolite concentrations reflecting the energy and the redox state in the tissue. Ten normal subjects and ten patients with peripheral vascular occlusive disease were investigated. 2. In normal subjects the pO2 at the end of exercise was related to the intensity of the exercise, expressed as effect (J/s) per contraction. 3. In both patients and normal subject the pO2 was related to the [ATP]/[ADP] ratio, the [lactate/[pyruvate] ratio and the phosphocreatine concentration in the muscle tissue at rest and during exercise. 4. At each pO2 value, a lower [lactate/[pyruvate] ratio was found in the muscle tissue of the patients compared with that of normal subjects. This was interpreted as a beneficial effect of the higher oxidative-enzyme capacity in the muscle of the patients. 5. The results show the importance of pO2 for the regulation of the energy and the redox state of the tissue. During exercise the changes induced in pO2 and thus the energy state will stimulate the respiratory rate. This might be an important link in triggering the oxidative-enzyme capacity in response to physical training as well as in peripheral vascular occlusive disease.     

Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity.     

Stimulation and partial stabilization of human histidyl-tRNA synthetase by hemoglobin

Histidyl-tRNA synthetase, an enzyme against which antibodies are directed in some patients with polymyositis, has been purified 5000-fold from HeLa cells, but was extremely labile to dilution or on storage at -80 degrees C. In order to facilitate study of the biochemical and immunological properties of the enzyme, a stabilizer was sought. Hemoglobin at 2 mg/ml was found to stimulate the enzyme and also partially preserved the activity of the enzyme stored at a low concentration (less than 10 micrograms/ml). Hematin, but not the globin protein, could substitute for hemoglobin in stimulating the enzyme.     

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., and Robert, C. H. (1987) Biochemistry, 26, 3995-4002), the heats for binding two and four oxygen molecules were found to be simple multiples of the first binding heat. A direct consequence of equal stepwise heats is invariance of the shape of the binding curve with temperature, as pointed out by Wyman (Wyman, J. (1939) J. Biol. Chem. 127, 581-599). Titration microcalorimetry was also performed for the binding of carbon monoxide to hemoglobin. While the tight binding of CO precludes high-precision binding measurements, it does allow one to accurately determine the heat of ligation as a function of the CO bound. In these titrations a uniform heat of reaction is not observed, but the heat of binding increases markedly near the end point. This implies that the stepwise binding enthalpy for adding the third CO molecule is anomalously endothermic and for adding the fourth strongly exothermic. A similar phenomenon cannot be ruled out in the case of oxygen because of imprecision intrinsic in the analysis of the weaker ligand binding.     

Scientific and technological aspects of oxygen-sensitive electrodes for measurements of oxygen partial pressure in patch-clamp experiments

Anoxia/hypoxia induces dramatic changes in brain activity leading to the damages in brain structure. Several minutes of decrease in environmental oxygen partial pressure (P(O2)) can irreversibly destroy nerve cells. Therefore, investigations of intracellular mechanisms responsible for hypoxia-induced changes of mammalian nerve system are very important. On-line adjustment and measurements of P(O2) in bath solution during patch-clamp experiments are especially topical. At the recent time, a special interest on the on-line measurements of oxygen contents in bath solution has appeared as a result of application of old approaches previously used for polarographic measurements of oxygen contents in the blood and tissues. Here we describe the simple method of manufacturing oxygen-sensitive microelectrodes, which can be used with standard patch-clamp amplifier. We also describe the main principles of polarographic method and properties of oxygen-sensitive electrodes used in patch-clamp experiments.