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Interactions of human alkaline phosphatase isoenzymes with triazine dyes using affinity partitioning, affinity chromatography and difference spectroscopy.

Aqueous two-phase systems consisting of dextran, polyethylene glycol and dye-liganded polyethylene glycol were employed to investigate the affinity partitioning behaviour of isoenzymes of human alkaline phosphatase. Whereas in the system without a dye ligand the partition coefficients of the isoenzymes from human intestine and placenta were identical, the isoenzyme from human liver showed a significantly lower partition coefficient under the same conditions. After addition of dye-liganded polyethylene glycol two groups of dyes possessing substantial affinities to the isoenzymes were found. One, represented by Procion Yellow HE-3G, interacts specifically with the active centre of the isoenzymes. Differences in the affinity of the isoenzymes towards the individual dye ligands are caused only by the carbohydrate content, especially by the terminal sialic acid residues. The other group of dye ligands, represented by Procion Navy MX-RB, binds obviously in a more complex fashion involving other binding sites, which are only present in alkaline phosphatase of human liver. Procion Navy MX-RB was found to function as a suitable affinity ligand for the separation of human liver alkaline phosphatase from the other isoenzymes. Differences in the primary structure of two allelic forms of human placental alkaline phosphatase [(SS) and (F)] are not recognized in aqueous two-phase systems with or without dye-liganded polyethylene glycol.

Alkaline Phosphatase↗

Synthesis and anti-HIV-1 activity of 4-[4-(4,6-bisphenylamino-triazin-2-ylamino)-5-methoxy-2-methylphenylazo]-5-hydroxynaphthalene-2,7-disulfonic acid and its derivatives.

A structure-based design approach has been used to optimize a lead HIV-1 entry inhibitor targeted to the envelope glycoprotein gp41. The docking study on this lead compound revealed important structural requirements that need to be preserved as well as structural non-requirements that could be eliminated to substantially reduce the molecular size of the lead compound. Based on the results from docking study, a limited number of analogues were designed and synthesized. This approach yielded a new analogue (compound 4) that retained the anti-HIV-1 activity with reduced molecular size approaching towards more drug-like character.

Anti-HIV Agents↗

Molecularly imprinted polymers for triazine herbicides prepared by multi-step swelling and polymerization method. Their application to the determination of methylthiotriazine herbicides in river water.

Uniformly-sized, molecularly imprinted polymers (MIPs) for atrazine, ametryn and irgarol were prepared by a multi-step swelling and polymerization method using ethylene glycol dimethacrylate as a cross-linker and methacrylic acid (MAA), 2-(trifluoromethyl) acrylic acid (TFMAA) or 4-vinylpyridine either as a functional monomer or not. The MIP for atrazine prepared using MAA showed good molecular recognition abilities for chlorotriazine herbicides, while the MIPs for ametryn and irgarol prepared using TFMAA showed excellent molecular recognition abilities for methylthiotriazine herbicides. A restricted access media-molecularly imprinted polymer (RAM-MIP) for irgarol was prepared followed by in situ hydrophilic surface modification using glycerol dimethacrylate and glycerol monomethacrylate as hydrophilic monomers. The RAM-MIP was applied to selective pretreatment and enrichment of methylthiotriazine herbicides, simetryn, ametryn and prometryn, in river water, followed by their separation and UV detection via column-switching HPLC. The calibration graphs of these compounds showed good linearity in the range of 50-500 pg/mL (r > 0.999) with a 100 mL loading of a river water sample. The quantitation limits of simetryn, ametryn and prometryn were 50 pg/mL, and the detection limits were 25 pg/mL. The recoveries of simetryn, ametryn and prometryn at 50 pg/mL were 101%, 95.6% and 95.1%, respectively. This method was successfully applied for the simultaneous determination of simetryn, ametryn and prometryn in river water.

Herbicides↗

Studies on the interaction of extended terpyridyl and triazine metal complexes with DNA.

The interaction with duplex DNA of a series of 12 previously synthesised ruthenium (II) complexes incorporating extended terpyridines or analogous ligands is reported. The interaction was investigated using absorption spectroscopy and also, when possible, luminescence spectroscopy. Binding parameters for the interactions were estimated using established models. It was found that binding affinities are relatively large and their magnitude is somewhat dependent on the metal centre attached to the ligand. Viscosity studies reveal that all the complexes interact with DNA through groove binding and not intercalation. The variation in binding affinities of these complexes is rationalised by a consideration of electrostatic and steric factors.

DNA↗

Promutagen activation of triazine herbicides metribuzin and ametryn through Vicia faba metabolism inducing sister chromatid exchanges in human lymphocytes in vitro and in V. faba root tip meristems.

The aim of our study was the induction of sister chromatid exchanges (SCE) in human lymphocytes in vitro and in root tip meristems of Vicia faba to evaluate the genotoxic effects of metribuzin and ametryn. Direct treatments of these herbicides on human lymphocytes in vitro applied 24 h after the beginning of culture did not induce SCE; however, they showed a cytotoxic effect in the cultures expressed as cellular death. On the contrary, when extracts of V. faba roots, treated for 4 h with metribuzin and ametryn (in vivo activation), were added to the lymphocyte cultures, SCEs were significantly induced with an asymptotic response. Negative responses appeared with the in vitro assays, in which metribuzin and ametryn were added directly to the 48 h lymphocyte cultures for 4 h. Nevertheless, in treatments in which the S10 metabolic mix was added, the SCE frequencies were significantly different to the control, although a concentration-response relationship was only observed with metribuzin. The results showed that both herbicides needed the V. faba metabolism to produce SCE in human lymphocyte cultures. Metribuzin and ametryn applied to V. faba root tip meristems for 4 h increased SCE frequency significantly, and a concentration-response relationship was observed with both herbicides.

Biotransformation↗

Fractionation of the ribosome inactivating protein preparations with triazine dyes.

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.

Antibodies, Monoclonal↗

Use of triazine dyes in the affinity chromatographic purification of alkaline phosphatase from calf intestine.

Dye-ligand chromatography was examined as a method for the purification of alkaline phosphatase (EC.3.1.3.1). Forty six dye-Matrex Gels were assessed for their ability to bind alkaline phosphatase. Most dye adsorbents bound significant quantities of the enzyme. Three dye columns were examined in more detail for their selectivity using gradients of potassium chloride to desorb enzyme protein. Purification of alkaline phosphatase using Cibacron blue 3GA-Sepharose 6B chromatography was enhanced by using affinity elution. The best purifications (290-fold) were obtained using pulsed elution with the substrate alpha-naphthyl phosphate although the inhibitor, inorganic phosphate, was also useful (128-fold purification).

Alkaline Phosphatase↗