Pilar Gutiérrez Navarro⁽¹⁾, Iluminada Hernández⁽²⁾, Pedro Martínez de las Parras⁽³⁾, Abdelkrim El Bekkouri⁽⁴⁾, Raquel García^(5)
(1)Facultad de Farmacia. Universidad de Granada. - Granada. Spain
⁽²⁾Departamento de Química Física.. Universidad de Granada - Granada. Spain
⁽³⁾Dpto. Química Física. Universidad de Granada - Granada. Spain
⁽⁴⁾⁽⁵⁾Dpto. Quimica Fisica. Universidad de Granada - Granada. Spain

		[Biophysics]
	[Biochemistry]

EXPERIMENTAL

Substances and reagents

Sodium ampicillin (99%) was purchased from Aldrich.. Anhydrous cadmium chloride, cobalt nitrate hexahidrate and manganese clroride tetrahidrate were obtained from Merck, Anhydrous zinc chloride from Sigma and methanol of over 99% purity was purchased from Merck.

Instrumentation

A Shimadzu ( Kyoto, Japan ) RF 5000 spectrofluorimeter equipped with a Julabo F 10 thermostat was used for fluorescence measurements . We also used a Perkin- Elmer Lambda 16 spectrofhotometer equipped with a Selecta thermostat (  0.1 ºC) and a computer equipped with UV Winlab software from Perkin- Elmer. All measurements were performed with 1cm thick spectrophotometric cells.

Kinetic measurements

The kinetic study was carried out from intensity of fluorescence and absorption measurements. All kinetic experiments were performed at 25º C in methanol.

The kinetic mixtures were prepared directly on the spectrophotometric cell. In all kinetic experiences, solutions of appropriate concentration were prepared to obtain the desired concentration on the cell. The most diluted reagent was pipetted and placed on the cell inside the spectophotometer. Once termal equilibrium was achieved, a 100 l volume of a solution of appropiate concentration of the other reagent was added.

The kinetic reaction of ampicillin catalysed by Cd²⁺ was studied in a n earlier work⁽¹²⁾ and was followed by the appearance of chromophore near 280 nm. The appearance of this absorption band was attributed to the breaking of the ß -lactam ring and the formation of the corresponding penamaldic derivative.

The degradation reaction in presense of Zn²⁺ have also followed by the appearance of the band at 282 nm, and the Co²⁺ and Mn²⁺ reactions changes absorption at 331 and 250 , respectively. In the kinetic study a series of methanolic solutions were assayed containing different excesses of ampicillin ( ranging from 1 to 12 x 10-4 M ) and constant metal concentration ( 1x 10-4 M ); and also in ampicillin solutions of constant concentration ( 1x10-4 M ) and different metal ion concentration ( ranging from 0 to 750 x 10-4 M). Initial rates ( Vobs ) of product appearance , in this case, were calculated in the same way as reported earlier12 for the reactión in the presence of Cd²⁺.

In the study corresconding to Co²⁺ and Mn²⁺ , the initial rate constants of ampicillin degradation were obtained by measuring the change the absorbance, at appopiate wavelengh, with respect to the total change.

The degradation reaction catalysed by Zn²⁺ were also monitored fluorimetrically at the relevant excitation and emision wavelengths. In the first times, one hour, the formation of a fluorescent product is observed for the reaction catalalysed by Zn²⁺ , whereas for the reactions catlatysed by other metal ions considered the appearance of fluorescence emission is also observed after 24 hours of reaction.

Excitation and emision spectra

In all experiments the fluorescence measurements were made at 25 º C .The uncorrected excitation and emission spectra corresponding to each of the systems considered were obtained from kinetic mixtures with each of the metal ions considered.

Discussion Board

Any Comment to this presentation?

[ABSTRACT] [INTRODUCTION] [EXPERIMENTAL] [RESULTS AND DISCUSSION] [FIGURES] [FIGURES-2] [REFERENCES] [Discussion Board]

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		[Biophysics]
	[Biochemistry]

Pilar Gutiérrez Navarro, Iluminada Hernández, Pedro Martínez de las Parras, Abdelkrim El Bekkouri, Raquel García
Copyright © 1999-2000. All rights reserved.

Last update: 11/02/00