Study of the interaction of pyridoxal-5'-phosphate and human serum albumin in the presence of copper ions by circular dichroism

When pyridoxal-5'-phosphate (PLP) binds with human serum albumin (HSA) in the molar ratio of 1:1, negative in sign induced Cotton effect is observed in the ligand absorption band. This high affinity center is localized in the second domain at an alpha-helical site of the protein molecule. As a result of adding Cu2+ equimolar concentration a new optically active PLP binding site with the positive sign of Cotton effect is acidic medium and the opposite one in alkaline medium is formed on Lys-4 at the beginning of the polypeptide chain. Inversion of the CD spectrum occurs over the same range of pH of the medium as the change in symmetry of electric field surroundings (rhombic in equilibrium axial) of the copper ion within the equimolar ternary complex HSA: PLP: Cu2+.     

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.     

Copper and nickel binding to canine serum albumin. A circular dichroism study

The binding of copper and nickel to canine serum albumin has been studied using circular dichroism. In the 320-700 nm region, only a single positive extremum was observed at about 664-667.5 nm for copper bound to canine serum albumin. The intensity of this extremum was found to increase until a Cu2+/albumin molar ratio of 3 was reached. Further addition of Cu2+ led to a decrease in ellipticity. The absence of any extrema in the 560-570 and 480-510 nm regions showed that histidines were not involved in copper binding to canine albumin. In the case of nickel, initial binding was found to take place at the N-terminal tripeptide. At higher nickel to albumin molar ratios, circular dichroism spectra indicated the presence sulfur containing ligands but showed no evidence for the involvement of histidines. Canine serum albumin was found to bind six or more Cu2+ and Ni2+ ions with affinities that are lower than for human or bovine serum albumin.     

Reactivity of Cu2(lonazolac)4, a lipophilic copper acetate derivative

An acetate-like copper complex of lonazolac (3-(p-chlorophenyl)-1-phenyl-pyrazole-4-acetate) was prepared and characterized. The copper of Cu₂-(lonazolac)₄ was spin-coupled and remained EPR-silent. Water and organic solvents did not affect this magnetic interaction. Superoxide dismutase activity of the Cu complex in micromolar concentrations was detectable in the presence of up to 900 μg per ml of serum albumin or whole serum protein. At 700 μM albumin concentration, a ternary complex between Cu₂(lonazolac)₄ and the protein was formed. The original acetate-copper coordination changed to a biuret-type copper bonding as seen from EPR and electron absorption spectrometry. Lonazolac did not induce a detectable conformational change of the protein near or at the copper binding site. Equilibrium dialysis and optical titration experiments revealed that essentially all copper of the Cu₂-(lonazolac)₄ complex was bound in the specific binding site of serum albumin. The copper complex proved to be an effective inhibitor of lipid peroxidation.     

On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin: effect of epsilon-NH(2) groups blocking and chloroform binding

Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when epsilon-NH(2) groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal epsilon-NH(2) groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between epsilon-NH(2) groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR-HSA complex.     

Metal ion-dependent binding of gastrin to albumin

Binding of 125I-[Nle15]gastrin to albumin purified from porcine serum, from porcine gastric mucosal cytosol, and from bovine serum has been demonstrated by covalent cross-linking and ultracentrifugation. Binding was enhanced in the presence of Zn2+, Ni2+, Cu2+, Co2+, and Cd2+, but not Ca2+, Mg2+, or Mn2+. The best fit to the binding data for bovine serum albumin was obtained with a model assuming two nonequivalent binding sites. The affinity of both sites for gastrin was increased in the presence of 100 microM Zn2+ or Ni2+ ions. The highest association constant observed was 2.3 X 10(5) M-1 in the presence of 100 microM Zn2+ ions. The similarity of the Zn(2+)-dependence of binding for bovine and porcine serum albumins, despite the replacement of His3 by Tyr, suggested that the N-terminal metal ion-binding site was not involved. Although all gastrin affinities were reduced by 50% in the presence of 150 mM NaCl, the Zn(2+)-dependence of binding was retained. We therefore propose that the ternary complex of gastrin, Zn2+ ions, and albumin may play a physiological role in the serum transport of Zn2+ ions and in the uptake of Zn2+ ions from the lumen of the gastrointestinal tract.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Copper catalyzed oxidation of ascorbate (vitamin C). Inhibitory effect of catalase, superoxide dismutase, serum proteins (ceruloplasmin, albumin, apotransferrin) and amino acids

The inhibitory effect of catalase and superoxide dismutase on copper catalyzed oxidation of ascorbate is probably due to a binding of copper ions. Scavengers of hydroxyl ions and singlet oxygen had no effect on the ascorbate oxidation rate. Copper binding serum proteins reduced the oxidation rate; the order of effectiveness being: Ceruloplasmin greater than human albumin = bovine albumin greater than apotransferrin. The excellent protection obtained with catalase and ceruloplasmin is possibly due to a strong affinity for cuprous ions generated during the reaction. Cupric ion binding amino acids (His, Thr, Glu, Gln, Tyr) had considerably weaker protective effect than the proteins studied. Apparently they do not compete favorably with ascorbate for cupric ions.     

Affinity labeling the DNA polymerase alpha complex. I. Pyridoxal 5'-phosphate inhibition of DNA polymerase and DNA primase activities of the DNA polymerase alpha complex from Drosophila melanogaster embryos

DNA polymerase alpha from Drosophila melanogaster embryos is a multisubunit enzyme complex which can exhibit DNA polymerase, 3'----5' exonuclease, and DNA primase activities. Pyridoxal 5'-phosphate (PLP) inhibition of DNA polymerase activity in this complex is time dependent and exhibits saturation kinetics. Inhibition can be reversed by incubation with an excess of a primary amine unless the PLP-enzyme conjugate is first reduced with NaBH4. These results indicate that PLP inhibition occurs via imine formation at a specific site(s) on the enzyme. Results from substrate protection experiments are most consistent with inhibition of DNA polymerase activity by PLP binding to either one of two sites. One site (PLP site 1) can be protected from PLP inhibition by any nucleoside triphosphate in the absence or presence of template-primer, suggesting that PLP site 1 defines a nucleotide-binding site which is important for DNA polymerase activity but which is distinct from the DNA polymerase active site. PLP also inhibits DNA primase activity of the DNA polymerase alpha complex, and primase activity can be protected from PLP inhibition by nucleotide alone, arguing that PLP site 1 lies within the DNA primase active site. The second inhibitory PLP-binding site (PLP site 2) is only protected from PLP inhibition when the enzyme is bound to both template-primer and correct dNTP in a stable ternary complex. Since binding of PLP at site 2 is mutually exclusive with template-directed dNTP binding at the DNA polymerase active site, PLP site 2 appears to define the dNTP binding domain of the active site. Results from initial velocity analysis of PLP inhibition argue that there is a rate-limiting step in the polymerization cycle during product release and/or translocation.     

Does cobalt really bind tightly to hemocyanin active sites?

The analysis of Co(II)-apoHc complexes of two arthropodan species (freshwater crayfish): Orconectes limosus and Astacus astacus enabled to reach some conclusions about possible cobalt binding sites in the hemocyanin molecules. The occurrence of binding sites for Co(II) at sites other than the active center has been demonstrated. We excluded the possibility of strong binding of EDTA-non-removable cobalt ions in the binding sites occupied by copper. There were no differences between apoHc and the Co(II)apoHc complex in terms of the amount of bound Cu(I) ions and the kinetics of Cu(I) ion reconstitution.Abbreviations He hemocyanin - apoHc apohemocyanin - oxyHc oxyhemocyanin - Co-Hc hemocyanin complex with cobalt ions Offprint requests to: E. Serafln     

Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin

The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots. Evidence for additional copper binding sites in Cp and bSA was obtained suggesting a role for copper ion in the homeostatic regulation of Cu(II) and other metal ions in the serum. In the binding studies the Cp was freed of exogenous Cu(II) by passing it over a Chelex-100 column. Two flow rates were used, 4 ml/hr and 40 ml/hr, which removed Cu(II) of different affinities. Cp passed at the slower flow rate (Cp4) only contained the prosthetic copper atoms. Cp passed at the faster flow rate (Cp40) contained one additional copper atom with a Ka approximately 10(7) M-1. Another 2-6 Cu(II) ion could be added to the Cp40 with an average affinity of about Ka approximately 10(5) M-1. The Cu(II) ions found in Cp provide two distinguishable classes: (1) the prosthetic copper atoms and (2) the exogenous copper atoms that can be removed by Chelex-100. For bSA one copper atom was bound strongly with a Ka value approaching 10(12) - 10(13) M-1 and was not removed by Chelex-100 at any flow rate. A second copper atom was found with a Ka = 5.2 x 10(6) M-1 and was removed by Chelex-100 at 4 ml/hr. Three additional copper atoms were bound with a Ka = 1.6 x 10(5) M-1; they were readily removed by Chelex-100 at 40 ml/hr but were nondialysable.     

Binding of zinc and cadmium to human serum albumin

1. The interaction of zinc and cadmium ion with human serum albumin (HSA) is evaluated and compared by potentiometric titration method and computer simulation of complex equilibria. 2. Zinc binds to histidine and free amino groups, cadmium in addition to basic functional groups of the protein. 3. Whereas zinc binds stronger in 1:1 complexes, chelate binding favours cadmium ions. 4. Within biological pH-conditions, high amounts Zn(II) and even more of Cd(II) will be bound to HSA.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

The formation and nature of the mixed valence copper-D-penicillamine-chloride cluster in aqueous solution and its relevance to the treatment of Wilson's disease

Complex formation between D-penicillamine (Pen) and copper(II) ions has been studied under simulated physiological conditions in both the presence and absence of the blood plasma constituents albumin, alanine, histidine, and zinc(II). Chromatographic and uv/vis and electron spin resonance (esr) spectroscopic methods were used. The major species formed, at neutral pH and 0.15 mol dm-3 NaCl, is the violet species which is shown to have the same stoichiometry as the recently reported solid-state complex, i.e., [Cu8I Cu6II (Pen)12 Cl] 5-. The rate of formation of this species (MVC) is shown to be dependent on the Cu concentration, Cu:Pen ratio, relative Cl- ion concentration, pH, and temperature. Formation is inhibited by the presence of O2 and biological chelates. At the concentration levels found in blood plasma it is unlikely that the MVC ion has any significance in the therapeutic action of penicillamine in the treatment of Wilson's disease. Reexamination of the aqueous Cu-albumin-pen system reinforces earlier findings that pen is unable to mobilize Cu that is bound to albumin. Significant binding of pen to the protein is observed is not related to any protein-bound copper ions. Evidence that ternary complexes of the type amino acid-Cu-Pen can form in blood plasma is presented. These are unlikely, however, to be physiologically significant and the copper depletion induced by Pen in Wilson's disease cases must be elsewhere than in the blood plasma compartment.     

Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase

Metal binding at the activating site of rabbit muscle phosphoglucomutase has been studied by 31P, 7Li, and 113Cd NMR spectroscopy. A 7Li NMR signal of the binary Li+ complex of the phosphoenzyme was not observed probably because of rapid transverse relaxation of the bound ion due to chemical exchange with free Li+. The phosphoenzyme-Li+-glucose 6-phosphate ternary complex is more stable, kinetically, and yields a well-resolved peak from bound Li+ at -0.24 ppm from LiCl with a line width of 5 Hz and a T1 relaxation time of 0.51 +/- 0.07 s at 78 MHz. When glucose 1-phosphate was bound, instead, the chemical shift of bound 7Li+ was -0.13 ppm; and in the Li+ complex of the dephosphoenzyme and glucose bisphosphate a partially broadened 7Li+ peak appeared at -0.08 ppm. Thus, the bound metal ion has a somewhat different environment in each of these three ternary complexes. The 113Cd NMR signal of the binary Cd2+ complex of the phosphoenzyme appears at 22 ppm relative to Cd(ClO4)2 with a line width of 20 Hz at 44.4 MHz. Binding of substrate and formation of the Cd2+ complex of the dephosphoenzyme and glucose bisphosphate broaden the 113Cd NMR signal to 70 Hz and shift it to 75 ppm. The 53 ppm downfield shift upon the addition of substrate along with 1H NMR data suggests that one oxygen ligand to Cd2+ in the binary complex is replaced by a nitrogen ligand at some intermediate point in the enzymic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that carnosine and anserine may participate in Wilson's disease

Carnosine, anserine and copper(II) ion all bind to specific sites on bovine serum albumin, and, in addition, both dipeptides chelate copper(II) ion in the absence of serum albumin. Thus a solution of dipeptide, copper(II) ion and serum albumin exhibits several complexes that arise from the competing binding reactions. Since a change in this complex equilibrium might occur in Wilson's disease, we have investigated the reactions between the various complexes with NMR and ESR spectroscopy. Serum albumin simultaneously binds the copper(II) ion and carnosine to separate sites rather than forming a mixed chelate, but carnosine still is capable of competing with serum albumin for subsaturating amounts of copper.     

Analysis of ESR spectra in Mn2+-plant adenylate kinase complex

Interaction of plant adenylate kinase with Mn2+-adenine nucleotide binary complex was studied by ESR technique at room temperature. The ligand environment of Mn2+ in the ternary Mn2+-adenine nucleotide-enzyme complex was shown to change, as a result of enzyme binding as compared with that of binary complex. These changes seem to be due to substitution of protein molecules for water and adenine nucleotide ones, coordinated to Mn2+ ion on ternary complex formation. The same results were obtained in ESR studies on rabbit muscle myokinase. This fact may be considered as an evidence, that plant adenylate kinase is identical to animal one in its interaction with adenine nucleotides and manganese ions.     

Metals in biochemistry : P.M. Harrison and R.J. Hoare,Chapman & Hall,New York, 1980, 80 pp. $5.95 (paper)

An ultrafiltration technique was used to study stripping by glycine of the first copper and zinc ion equivalents bound by bovine, dog, and rat serum albumins at pH 7.5. Affinity of dog serum albumin for copper was poorer than for the other albumins, consistent with the absence in the former albumin of the copper binding site present at the amino terminus of the latter albumins. Affinities of all three proteins for zinc were similar, suggesting that the albumin amino terminus is not the primary zinc ion binding site.