Peroxidase activity of cationic metalloporphyrin-antibody complexes

Peroxidase activity of a complex of water-soluble cationic metalloporphyrin with anti-cationic porphyrin antibody is reported. Antibody 12E11G, which was prepared by immunization with a conjugate of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridyl)porphine iodide (3MPy1C), bound to tetramethylpyridylporphyrin iron complex (FeIII-TMPyP) with the dissociation constant of 2.6 x 10(-7) M. The complex of antibody 12E11G with FeIII-TMPyP catalyzed oxidation of pyrogallol, catechol, and guaiacol. A Lineweaver-Burk plot for the oxidation of pyrogallol catalyzed by the FeIII-TMPyP-antibody complex showed Km=8.6 mM and kcat=680 min(-1). Under the same conditions, Km and kcat for horseradish peroxidase (HRP) were 0.8 mM and 1750 min(-1), respectively. Although the binding interaction of the antibody to the substrates was one order lower than that of native HRP, the peroxidase activity of this system was in the same order of magnitude as that of HRP.     

Mimicking protein-protein electron transfer: voltammetry of Pseudomonas aeruginosa azurin and the Thermus thermophilus Cu(A) domain at omega-derivatized self-assembled-monolayer gold electrodes

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.6) and subunit II (Cu(A) domain) of ba(3)-type cytochrome c oxidase (T. thermophilus) (pI = 6.0) exhibit optimal voltammetric responses on 1:1 mixtures of [H(3)C(CH(2))(n)()SH + HO(CH(2))(n)()SH] SAMs. The electron transfer (ET) rate vs distance behavior of azurin and Cu(A) is independent of the omega-derivatized alkanethiol SAM headgroups. Strikingly, only wild-type azurin and mutants containing Trp48 give voltammetric responses: based on modeling, we suggest that electronic coupling with the SAM headgroup (H(3)C- and/or HO-) occurs at the Asn47 side chain carbonyl oxygen and that an Asn47-Cys112 hydrogen bond promotes intramolecular ET to the copper. Inspection of models also indicates that the Cu(A) domain of ba(3)-type cytochrome c oxidase is coupled to the SAM headgroup (H(3)C- and/or HO-) near the main chain carbonyl oxygen of Cys153 and that Phe88 (analogous to Trp143 in subunit II of cytochrome c oxidase from R. sphaeroides) is not involved in the dominant tunneling pathway. Our work suggests that hydrogen bonds from hydroxyl or other proton-donor groups to carbonyl oxygens potentially can facilitate intermolecular ET between physiological redox partners.     

FTIR detection of protonation/deprotonation of key carboxyl side chains caused by redox change of the Cu(A)-heme a moiety and ligand dissociation from the heme a3-Cu(B) center of bovine heart cytochrome c oxidase

FTIR spectral changes of bovine cytochrome c oxidase (CcO) upon ligand dissociation from heme a(3)() and redox change of the Cu(A)-heme a moiety (Cu(A)Fe(a)()) were investigated. In a photosteady state under CW laser illumination at 590 nm to carbonmonoxy CcO (CcO-CO), the C-O stretching bands due to Fe(a3)()(2+)CO and Cu(B)(1+)CO were identified at 1963 and 2063 cm(-)(1), respectively, for the fully reduced (FR) state [(Cu(A)Fe(a)())(3+)Fe(a3)()(2+)Cu(B)(1+)] and at 1965 and 2061 cm(-)(1) for the mixed valence (MV) state [(Cu(A)Fe(a)())(5+)Fe(a3)()(2+)Cu(B)(1+)] in H(2)O as well as in D(2)O. For the MV state, however, another band due to Cu(B)(1+)CO was found at 2040 cm(-)(1), which was distinct from the alpha/beta conformers in the spectral behaviors, and therefore was assigned to the (Cu(A)Fe(a)())(4+)Fe(a3)()(3+)Cu(B)(1+)CO generated by back electron transfer. The FR-minus-oxidized difference spectrum in the carboxyl stretching region provided two negative bands at 1749 and 1737 cm(-)(1) in H(2)O, which were apparently merged into a single band with a band center at 1741 cm(-)(1) in D(2)O. Comparison of these spectra with those of bacterial enzymes suggests that the 1749 and 1737 cm(-)(1) bands are due to COOH groups of Glu242 and Asp51, respectively. A similar difference spectrum of the carboxyl stretching region was also obtained between (Cu(A)Fe(a)())(3+)Fe(a3)()(2+)Cu(B)(1+)CO and (Cu(A)Fe(a)())(5+)Fe(a3)()(2+)Cu(B)(1+)CO. The results indicate that an oxidation state of the (Cu(A)Fe(a)()) moiety determines the carboxyl stretching spectra. On the other hand, CO-dissociated minus CO-bound difference spectra in the FR state gave rise to a positive and a negative peaks at 1749 and 1741 cm(-)(1), respectively, in H(2)O, but mainly a negative peak at 1735 cm(-)(1) in D(2)O. It was confirmed that the absence of a positive peak is not caused by slow deuteration of protein. The corresponding difference spectrum in the MV state showed a significantly weaker positive peak at 1749 cm(-)(1) and an intense negative peak at 1741 cm(-)(1) (1737 cm(-)(1) in D(2)O). The spectral difference between the FR and MV states is explained satisfactorily by the spectral change induced by the electron back flow upon CO dissociation as described above. Thus, the changes of carboxyl stretching bands induced both by oxidation of (Cu(A)Fe(a)()) and dissociation of CO appear at similar frequencies ( approximately 1749 cm(-)(1)) but are ascribed to different carboxyl side chains.     

Strain-Induced “Band Flips” in Cyclodecaamylose and Higher Homologues

The cavity of the larger molecule has less space for guests! Unlike the structure of the smaller annular cyclodextrins, that of the higher homologues of cycloamyloses (CAs) with more than ten glucose units contains a 90° kink between adjacent glucose residues within one half of the molecule and a 180° band flip between adjacent units in different halves (see depicted section of the CA14 structure) to yield butterfly-shaped structures with narrow, groovelike cavities.     

Dynamic analysis of aggregation of methylene blue with polarized optical waveguide spectroscopy

Polarized optical waveguide (POW) spectroscopy permits analysis of the changing molecular state of methylene blue (MB), including aggregate order and orientation at the waveguide surface. Monomer or dimer, dissolved randomly in MB aqueous solution, tends to aggregate at the waveguide surface during air drying. Furthermore, POW spectroscopy dynamically revealed that MB molecules became oriented vertically to the waveguide surface with increasing aggregation order.     

Fluorescent high-performance liquid chromatographic determination of primary aromatic amines by formation of Schiff base

Summary The precolumn derivatization of several primary aromatic amines, especially phenolamines, with 1-pyrenealdehyde, and their determination by high-performance liquid chromatography with fluorescence detection have been studied. The derivatization proceeded in methanol to form the Schiff bases, which were chromatographed on the ODS column. The detection limit is 1–2 pmol. 1-Pyrenealdehyde was evaluated as a derivatization reagent for the selective analysis of phenolamines such as 2-phenylethylamine, in biogenic aromatic amines.     

A sensitive two-phase titration of anionic surfactants with a dilute tetraphenylphosphonium chloride solution

A visual indicator method is described for titrations of anionic surfactants with 5 × 10^-5 M tetraphenylphosphonium solution. Two dyes, the hydrophilic neutral red and the hydrophobic tetrabromophenolphthalein ethyl ester, are used as indicator in the presence of 1,2-dichloroethane.     

Preparation of cyclosophoraose-A and its complex-forming ability

Cyclosophoraoses (CySs) are unbranched cyclic (12)--D-glucans produced by many strains ofAgrobacterium andRhizobium. Pure CyS-A, the group member having the smallest molecular size (degree of polymerization 17), was efficiently prepared by liquid chromatography using charcoal and ODS columns from the culture fluid of the mutant strain RA-12 fromR. phaseoli AHU 1133. The complex-forming ability of CyS-A was estimated from its enhancement of the solubilities of slightly soluble guest molecules in water using methods [I], [II], and [III]. In [I], an aqueous solution of CyS-A was shaken with the guest molecule, while, in [II], it was shaken with an acetone solution of the guest compound. In method [III], freeze-dried CyS-A powder was stirred with an acetone solution of the guest compound. The CyS-A cavity is thought to be able to accommodate three-dimensionally extended guest molecules,e.g., indomethacin. Method [II] was the best for obtaining CyS-A inclusion complexes, while method [III] would be recommended if the guest molecule is labile in the presence of water. Crystalline CyS-A inclusion complexes have not been obtained, but CyS-A complexes are expected to greatly enhance the solubilities of slightly soluble or insoluble guest molecules in water, because CyS-A is much more soluble than -cyclodextrin. Method [II] or [III] may afford a useful means of obtaining oily drug,e. g., vitamin E and K₁, in an amorphous state.     

Evaluation of scopadulciol-related molecules for their stimulatory effect on the cytotoxicity of acyclovir and ganciclovir against Herpes simplex virus type 1 thymidine kinase gene-transfected HeLa cells

Herpes simplex virus type 1 thymidine kinase (HSV TK) is involved in both antiherpetic therapy and cancer gene therapy with acyclovir (ACV) and ganciclovir (GCV). Enhanced sensitivity to these drugs is advantageous in their clinical use. In the present study, scopadulciol (SDC) and its related compounds were evaluated for their stimulatory effect on the cytotoxicity of ACV and GCV by determination of selective toxicities against HSV TK-expressing HeLa cells. Although SDC remarkably potenciated the cytotoxicity of ACV and GCV, the other tested compounds showed only weak selectivity, except for compound 34.     

Copper(I) tert-butoxide-promoted 1,4 C sp 2-to-O silyl migration: generation of vinyl copper equivalents and their stereospecific cross-coupling with allylic,aryl, and vinylic halides

Successive treatment of the (Z)-gamma-trimethylsilyl allylic alcohols with copper(I) tert-butoxide and allylic halides followed by the tetrabutylammonium fluoride-assisted hydrolysis produced the allylation products, 2,5-alkadien-1-ols, with complete retention of configuration. Similar treatment of the organometallic intermediates with aryl and vinylic halides in the presence of palladium(0) catalyst gave the corresponding cross-coupling products in good yields. The stereoselective preparation of the starting materials is also described.