Junzo Hirose

Photoreversible antigen—antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K=5 × 107 M−1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.

The pH dependence of apparent binding constants between apo-superoxide dismutase and cupric ions

Abstract Apo-superoxide dismutase can bind four copper ions to two native copper- and two native zinc-binding sites. Four, stepwise, apparent binding constants of copper ions to the apo-enzyme were obtained from the equilibrium dialysis data at various pH values. These binding constants are pH dependent. In the low pH region, the binding of copper ions to the native copper binding sites has much larger apparent binding constants than to the native zinc sites. But the difference between apparent binding constants of copper ions to the native copper sites and those to the native zinc sites decreased with an increase of pH. At pH 10.0, it is impossible to distinguish between the binding of copper ions to the native copper sites and to the native zinc sites. The relationship between logarithm of apparent binding constants of copper sites and pH was linear with a slope of 2 in the pH region from 4.0 to 7.0. Above pH 7.0, apparent binding constants at the copper sites were almost pH independent. These results indicate that copper ions compete with two protons in the native copper-binding sites and that p K a values of the protons were almost 7. From apparent binding constants of copper ions to the apo-enzyme at pH 7.0 and 10.0, theoretical EPR-detectable copper contents were calculated and compared with EPR data obtained by J. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard ( Proc. Nat. Acad. Sci. USA , 1979 , 76 , 4245–4249).

Metal Preferences of Zinc-Binding Motif on Metalloproteases

Almost all naturally occurring metalloproteases are monozinc enzymes. The zinc in any number of zinc metalloproteases has been substituted by some other divalent cation. Almost all Co(II)- or Mn(II)-substituted enzymes maintain the catalytic activity of their zinc counterparts. However, in the case of Cu(II) substitution of zinc proteases, a great number of enzymes are not active, for example, thermolysin, carboxypeptidase A, endopeptidase from Lactococcus lactis, or aminopeptidase B, while some do have catalytic activity, for example, astacin (37%) and DPP III (100%). Based on structural studies of various metal-substituted enzymes, for example, thermolysin, astacin, aminopeptidase B, dipeptidyl peptidase (DPP) III, and del-DPP III, the metal coordination geometries of both active and inactive Cu(II)-substituted enzymes are shown to be the same as those of the wild-type Zn(II) enzymes. Therefore, the enzyme activity of a copper-ion-substituted zinc metalloprotease may depend on the flexibility of catalytic domain.

Measurement of apparent binding constant between copper ion and apo-bovine superoxide dismutase

The binding constants of copper ions to apo-bovine superoxide dismutase were measured by the method of equilibrium dialysis. The binding constant (108.9 M−1) of copper ion to the native copper site was much larger (106 times) than that to the native zinc site at pH 4.0. The two native binding sites of copper ions are identical and show no interaction in the measurement of the binding constants. The binding of copper ions to the native zinc sites involved release of two protons and competition between these two protons and copper ion was governed by the pH dependence of copper binding constant to the native zinc sites.

Inhibition of bovine carbonic anhydrase by 5-methyl-1,10-phenanthroline. Direct spectrophotometric evidence for ternary complex between the enzyme and chelating agent.

Abstract For the removal of the cobalt ion from cobalt-bovine carbonic anhydrase with 5-methyl-1,10-phenanthroline, it was proved spectrophotometrically that the substitution mechanism proceeded through the ternary complex in which the central metal ion bound with the protein and the chelating agent. The spectrum of the ternary complex had low molar absorption coefficient in visible region, so that it was assumed that the ternary complex had a five- or six-coordination geometry.

Coordination chemical studies on metalloenzymes: Measurement of binding constant between apo-tyrosinase and copper ion

Abstract The behavior of complexing agents for the copper removal reaction was studied by the equilibrium dialysis method. In the copper removal reaction, complexing agents are divided into two types: those that are reducing agents and those that are not. Sodium cyanide and sodium thiosulfate are of the first type, and 8-hydroxyquinoline-5-sulfonic acid, 2,2′-bipyridyl, and picolinic acid are of the second type. From equilibrium dialysis with the first type of complexing agent, the apparent binding constant (pH 6.0) between cuprous ions and apotyrosinase was calculated to be 10 15 m −1 . Similarly, the apparent binding constant (pH 6.0) between cupric ions and apo-tyrosinase was about 10 13 m −1 , which was calculated from equilibrium dialysis with the second type of complexing agent. The apparent binding constant between cuprous ions and apo-tyrosinase was larger than that between cupric ions and apo-tyrosinase.

Metal coordination geometry of ternary complex between cobalt-bovine carbonic anhydrase and multidentate ligands

Interaction of cobalt(II) bovine carbonic anhydrase with 3- and 4-pyridinecarboxylates, 2-pyridinecarboxylate, and 2,6-pyridinedicarboxylate has been investigated by the spectrophotometric method. The apparent formation constant of the ternary complex (ligand : cobalt ion : apoenzyme = 1 : 1 : 1) was determined from spectral data. The spectroscopic data of the ternary complex indicate that the 3- or 4-pyridinecarboxylate adduct has a five-coordination geometry through three donor atoms of the protein part of the enzyme, the carboxyl group of 3- or 4-pyridinecarboxylate, and a water molecule. 3- or 4-Pyridinecarboxylate behaves as a monodentate ligand. The spectrum of the ternary complex of 2-pyridinecarboxylate was very different from that of 3- or 4-pyridinecarboxylate. The spectra data indicate that 2-pyridinecarboxylate adduct has a five-coordination geometry and that it behaves as a bidentate ligand. The ternary complex of 2,6-pyridinedicarboxylate was so unstable that the spectrum of the ternary complex was determined by the indirect method. The spectrum of 2,6-pyridinedicarboxylate adduct shows lower molar absorption than that of 2-pyridinecarboxylate adduct. This result indicates that 2,6-pyridine dicarboxylate behaves possibly as a tridentate ligand.

Coordination chemical studies on metalloenzymes. IX. Properties of the ternary complex between cobalt(II)-bovine carbonic anhydrase and bidentate ligands

The spectrum, thermodynamic parameters, and proton longitudinal relaxation time of the ternary complex between various bidentate ligands (2-pyridinecarboxylate, 2-quinolincarboxylate, 8-quinolinecarboxylate, and 2-pyridylacetate) and cobalt(II)-bovine carbonic anhydrase were measured to clarify the nature of the ternary complex. The formation constants of the ternary complexes of bidentate ligands are in the order of (2-pyridinecarboxylate approximately greater than 8-quinolinecarboxylate much greater than 2-quinolinecarboxylate approximately equal to 2-pyridylacetate). The degree of the shift of the band characteristic of five-coordinate species at 13-15 kcm-1 (cm-1 X 10(-3)) and that of the higher energy band at 21-22 kcm-1 decrease almost in the same order. These results are explained on the basis of the contribution of the bond formation between the nitrogen atom of the heterocyclic ring of ligands and cobalt ion. The formation constants of the ternary complex of bidentate ligands were compared to the stability constants of various ligands with a cobalt ion but there is no correlation in these values. The rate constant of break-up of the ternary complex was discussed on the coordination geometry of the ternary complex on the basis of the degree of the distortion.

Antibodies against (1R,2R)-cyclohexanediamineplatinum(II)-DNA adduct recognize the conformational differences of isomeric analogues of cyclohexanediamine

Antibodies reactive to (1R,2R)-cyclohexanediamineplatinum(II)-DNA ((1R,2R)-cyclohexanediamine: 1R,2R-dach) adducts were elicited by immunization of rabbit with calf thymus DNA modified by Pt(1R,2R-dach)Cl2 at a ratio of bound platinum per nucleotide ((D/N)b) of 0.0335. In an enzyme-linked immunosorbent assay (ELISA), the binding of specific antibodies to Pt(1R,2R-dach)-DNA adduct (60 microliters of 1.235 x 10(-7) M Pt in each wells) on the assay plate was competitively inhibited by Pt(1R,2R-dach)-DNA adduct ((D/N)b = 0.0653) in the solution. Almost equal inhibition was observed with Pt(1S,2S-dach)-DNA ((D/N)b = 0.0412), an optical isomer of 1R,2R-dach. Pt(1R,2S-dach)-DNA ((D/N)b = 0.0371) and Pt(1R,3S-dach)-DNA ((D/N)b = 0.0281) in which the cyclohexane ring is stereochemically perpendicular to the platinum chelate plane, also inhibited antibody binding, but these adducts gave only incomplete inhibition at higher Pt-DNA adduct concentrations. Although Pt(1R,2R-dach)-d(GpG) and Pt(1R,2R-dach)(NH3)2 inhibited antibody binding, the affinity of the antibody for Pt(1R,2R-dach)(NH3)2 was lower than with Pt(1R,2R-dach)-DNA, and the inhibition behavior of Pt(1R,2R-dach)-d(GpG) was biphasic, i.e., at the lower concentration the inhibition curve was consistent with that of Pt(1R,2R-dach)-DNA, but at the higher concentration it shifted to that of Pt(1R,2R-dach)(NH3)2. The affinity of the antibody for cis-DDP was markedly lower than with Pt(1R,2R-dach)(NH3)2. These facts suggest that the antibodies may bind to the substituents (the platinum and its surroundings) of the various Pt complexes rather than the DNA structure altered by platinum binding.

A 1H NMR study of cobalt(II) arsanilazocarboxypeptidase A.

Cobalt(II) arsanilazotyrosine-248 carboxypeptidase A has been characterized through 1H NMR spectroscopy. The ability of the azoenzyme to form binary and ternary complexes with L- and D-phenylalanine and azide has been investigated. Comparison with the 1H NMR results obtained on unmodified cobalt(II) carboxypeptidase provides direct information on the specific effect of the presence of the azo group on the reactivity of the system. Marked differences in the interaction with D-phenylalanine have been observed, and structural inferences are drawn.