Jean-Claude Chottard

5-Lipoxygenase from potato tubers. Improved purification and physicochemical characteristics

Abstract Potato tubers are shown to contain at least three lipoxygenase isoenzymes. A very efficient extraction of lipoxygenase activity is obtained when a non-ionic detergent (0.1% Brij 99) is added to the homogenization buffer. The major isoenzyme, L 1 , has been purified in an almost homogeneous form with a good yield (18%) and a high specific activity (140–160 units/mg). It is efficiently stabilized by glycerol (20%, v/v). The purified L 1 isoenzyme is slightly contaminated by an 11-lipoxygenase, both having very close p I values (4.94 and 4.99, respectively). L 1 is a monomeric protein of M r 92 000 containing one iron atom per molecule. The native enzyme is in a pseudo-axial high-spin ferric state as indicated by EPR. Acting on linoleic acid, L 1 forms 9-hydroperoxyoctadecadienoic acid (9-HPOD) almost exclusively. With arachidonic acid, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) is the major product (70–75%) beside small amounts of 8(ifS)- and 9-HPETE. Due to the contaminating activity, 11-HPETE (15%) is also present. Formation of both 8( S )-HPETE and leukotriene A 4 hydrolysis products accounts for the intrinsic 8-lipoxygenase activity of the L 1 isoenzyme.

Second-order globalisation for the determination of activation parameters in kinetics

Abstract First order global analysis consists of linking common parameters across series of measurements, e.g., reaction kinetics measured at different wavelengths where the rate constants are the same for all kinetic traces at individual wavelengths. This approach is taken a step further in second order globalisation. A series of measurements is linked together by a new superimposed model which encompasses the individual measurements. The mathematics for the non-linear least-squares fit of the global parameters is presented. Two modes are possible depending on whether the linear parameters (absorption spectra) are constant or changing across the series. Factor analysis is incorporated for multivariate measurements. The procedures are exemplified with applications of activation analysis in chemical kinetics. Global analysis of complete temperature and pressure dependences results directly in the activation parameters of interest, i.e., activation enthalpies, entropies and volumes. Due to a significant decrease in the number of parameters to be fitted, the robustness is considerably improved.

A Complete Kinetic Study of GG versus AG Platination Suggests That the Doubly Aquated Derivatives of Cisplatin Are the Actual DNA Binding Species

The hairpin-stabilized double-stranded oligonucleotides d(TATGGTATT4ATACCATA) (I) and d(TATAGTATT4ATACTATA) (II) were allowed to react with the three aquated forms of the antitumor drug cisplatin (cis-[PtCl2(NH3)2], 1) which are likely candidates for DNA binding, that is, cis-[PtC1(NH3)2(H2O)]+ (2), cis-[Pt(NH3)2(H2O)2]2+ (3), and its conjugate base cis-[Pt(OH)(NH3)2(H2O)]+ (4). The reaction between I and [Pt(NH3)3(H2O)]2+ (5) was also studied for comparison. All reactions were monitored by HPLC. The platination reactions of I and II were carried out in NaClO4 (0.1M) at 293 K and at a constant pH of 4.5 +/- 0.1 for 2, 3, and 5. The data relative to the platination by 4 were obtained from measurements in unbuffered NaClO4 solutions (0.1M) at a starting pH close to neutrality, where 3 and 4 are present in equilibrium. In this case, a fit function describing the pH-time curve allowed the determination of the actual concentrations of 3, 4, and the dihydroxo complex. The platination rate constants characterizing the bimolecular reactions between either I or II and 2, 3, and 4 were individually determined along with the rate constants for hydrolysis of the chloro-monoadducts and for the chelation reactions of the aqua-monoadducts. The reactivity of compounds 2-5, which have the general formula cis-[Pt(NH3)2(H2O)(Y)]2+/-, decreases in the order 3>4>5>>2, that is, Y= H2O > OH- >NH3 >> Cl-, which is the order of decreasing hydrogen-bond donating ability of Y. Deprotonation of 3 to 4 reduces the reactivity of the platinum complex only by a factor of approximately equals 2, and both complexes discriminate between the different purines of I and II in the same manner. Whereas 3 and 4 react approximately three times faster with the GG sequence of I than with the AG sequence of II, 2 shows a similar reactivity towards both sequences. In view of the well-established preferential binding of cisplatin to GG sequences of DNA in vivo and in vitro, this result suggests that the actual DNA platination species are derived from double hydrolysis of cisplatin.

Kinetic aspects of interactions between DNA and platinum complexes

Abstract Kinetic studies on reactions between oligonucleotides and platinum complexes related to the antitumor drug cisplatin are described. These studies were motivated by the sequence-selectivity observed for the reaction between cisplatin and DNA. Comparison of oligonucleotide platination rate constants measured for the three complexes cis -[PtCl(NH 3 ) 2 (H 2 O)] + , cis -[Pt(NH 3 ) 2 (H 2 O) 2 ] 2+ , and [Pt(NH 3 ) 3 (H 2 O)] 2+ suggests that the hydrogen bond donating capacity of the platinum ligands enhances the platination rate. The binding preferences of cis -[Pt(NH 3 ) 2 (H 2 O) 2 ] 2+ and cis -[PtCl(NH 3 ) 2 (H 2 O)] + indicate that the latter species is unlikely to be the major species interacting with DNA in vivo. This conjecture is corroborated by reactivity and availability considerations. We also address the sequence-dependence observed for the hydrolysis of chloro-monoadducts and for the conversion of aqua-monoadducts to diadducts, and discuss possible mechanisms.

Chemical Reactions Between Copper Pigments and Oleoresinous Media

Abstract study demonstrates that resin and fatty acids are able to extract copper(II) ions from verdigris (copper acetate) and verditer (basic copper carbonate). Ligand exchange reactions of basic copper carbonate with fatty acids and resin acids are much slower than is the case with copper acetate. The browning of paint layers is closely correlated with the relative ease of copper extraction; the copper diffuses in the form of fatty acid or resin carboxylic acid complexes. These complexes are formed in the painting layer during grinding of the pigment with binding media containing oleoresin acids, as well as being formed at the interface of this layer and organic upper layers, such as varnishes.

Cobalt(III) Complexes with Carboxamido‐N and Sulfenato‐S or Sulfinato‐S Ligands Suggest that a Coordinated Sulfenate‐S is Essential for the Catalytic Activity of Nitrile Hydratases

A bis(sulfenato-S)CoIII complex and its corresponding bis(sulfinato-S) complex were prepared. Their catalytic nitrile hydration activity was tested. Whereas the former catalyzes the hydration of CH3CN under mild acidic conditions, the latter is inactive. A mechanism for this reaction is proposed, in relation with nitrile hydratase activity.

A Pentacoordinated Di-N-carboxamido-dithiolato-O-sulfinato-iron(III) Complex Related to the Metal Site of Nitrile Hydratase.

Postcoordination oxidation by dioxygen of one of the thiolate groups in a pentadentate N(2)S(3) ligand results in an iron(III) complex with two N-carboxamido, two thiolato, and one O-sulfinato ligands (see the CAMERON representation). This novel mixed coordination is similar to that determined for the inactive form of the nitrile hydratase from Rhodococcus sp. N-771, but differs by the O versus S binding of the sulfinato ligand.

Implication of 195Pt chemical shift anisotropy relaxation in n.m.r. studies of platinum complexes

A dominant chemical shift anisotropy relaxation mechanism can account, at high magnetic fields, for the disappearance of some of the 195Pt–1H and 195Pt–13C coupling constants and for the line broadening observed in the 195Pt n.m.r. spectra of PtII complexes.

Structural and spectroscopic characterization of a five-coordinate {Fe(NO)}6 complex derived from an iron complex with carboxamido N and thiolato S donors

Abstract An iron(III) complex, [Fe(N2S2)CI](Et4N)2 (1) with a mixed carboxamido nitrogen and thiolato sulfur donor set derived from (2-mercapto-isobutyryl)-o-phenylene diamine was prepared. The iron(III) is in a square pyramidal ligand environment. EPR, Mossbauer and variable-temperature susceptibility establishes that 1 possesses an S=3/2 ground state. The iron(III) oxidation state is stable over a 1.5 V range (Epc[Fe(N2S2)Cl]3−/2− =−1290 mV and Epa [Fe(N2S2)Cl]2−/1−=+220 mV versus SCE in CH3CN). Complex 1 reacted with a stoichiometric amount of NO yielding an air stable [Fe(N2S2)(NO)](Et4N) complex 2 which belongs, as does the [Fe(S2C4N2)2(NO](Et4N) nitrosyl dithiotene complex 4 to the {Fe(NO)}6 group. Both complexes have a square pyramidal structure with a linear FeNO moiety and a very short FeN(O) bond distance of 1.633 (2) and 1.612 A (4). They differ by the position of the νNO stretching frequency located at 1780 cm−1 for 2 and 1840 cm−1 for 4. Magnetic susceptibility measurements indicated the diamagnetic nature of both complexes. The Mossbauer spectra of complexes 1, 2, 3 ([Fe(S2C4N2)2]2(Na)2), and 4 consist of a doublet with a large quadrupole splitting in the case of 1 and 2 (3.278 and 3.149 mm s−1, respectively, at 293 K). Isomer shifts values of the nitrosylated compounds are lower (−0.171 for 2 vs. +0.177 for 1 at 293 K and +0.053 mm s−1 for 4 vs. +0.331 mm s−1 for 3 at 100 K).

Fast Interstrand Cross‐linking of Cisplatin–DNA Monoadducts Compared with Intrastrand Chelation: A Kinetic Study Using Hairpin‐Stabilized Duplex Oligonucleotides

The antitumor drug cisplatin forms two kinds of guanine-guanine cross-links with DNA: intrastrand, occurring mainly at GG sites, and interstrand, formed at GC sites. The former are generally more abundant than the latter, at least in experiments with linear duplex DNA. The formation of interstrand cross-links requires partial disruption of the Watson-Crick base pairing, and one could therefore expect the cross-linking reaction to be rather slow. In contrast with this expectation, kinetic measurements reported here indicate that interstrand cross-linking is as fast as intrastrand, or even faster. We have investigated the reactions between two hairpin-stabilized DNA duplexes, containing either a d(TGCA)(2) sequence (duplex TGCA) or a d(G(1)G(2)CA)-d(TG(3)CC) sequence (duplex GGCA), and the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), in an unbuffered solution kept at pH 4.5 +/- 0.1 and 20 degrees C. Using HPLC as the analytical method, we have determined the platination (first step) and chelation (second step) rate constants for these reaction systems. Duplex TGCA, in which the two guanines are quasi-equivalent, is found to be platinated very slowly (k=0.5 +/- 0.1M(-1)s(-1)) and to form the final interstrand cross-link very rapidly (k=13 +/- 3 x 10(-3) s(-11)). For GGCA, we find that G(1) is platinated rapidly (k=32 +/- 5M(-1)s(-1)) to form a long-lived monoadduct, which is only slowly chelated (k=0.039 +/- 0.001 x 10(-3) s(-1)) by G(2) (intrastrand), while G(2) is platinated one order of magnitude more slowly than G(1) (k=2.0 +/- 0.5M(-1)s(-1)) and chelated fairly rapidly both by G(1) (intrastrand: k=0.4 +/-0.1 x 10(-3) s(-1)) and G(3) (interstrand: k=0.2 +/- 0.1 x 10(-3) s(-1)); finally, G(3) is platinated at about the same rate as G(2) (k=2.4 +/- 0.5M(-1)s(-1)) and chelated very rapidly by G(2) (interstrand: k=10 +/- 4 x 10(-3) s(-1)). These results suggest that the low occurrence of interstrand cross-links in cisplatinated DNA is due to an extremely slow initial platination of guanines involved in d(GC)(2) sequences, rather than to a slow cross-linking reaction.