Affinity and stability modifications of immobilized alcohol dehydrogenase through multipoint copolymerization.

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), a potentially useful enzyme for cofactor regeneration processes, was covalently immobilized in a multipoint fashion by activation with acryloyl chloride and subsequent copolymerization in a polyacrylamide gel. Several properties such as the activity and stability were systematically studied for the free enzyme, the acryloate-enzyme and the immobilized enzyme. The activation energy was significantly lowered upon immobilization. The thermal stability of the immobilized enzyme was, however, greatly increased. But its maximum activity was observed at a lower temperature. These results suggest an important effect of the diffusional restrictions and of the mode of activation and immobilization on the activity and the stability of the enzyme.

Simple-kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl-chloride-activated agarose.

Yeast alcohol dehydrogenase was successfully immobilized on tresyl-chloride-activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%. Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this immobilized alcohol dehydrogenase.

Effect of the microenvironment on the kinetic properties of immobilized enzymes.

A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co-polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages. The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions. The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.

[Intracytoplasmic injection and embryo quality: comparison with conventional in vitro fertilization]. / Injection intracytoplasmique et qualité embryonnaire: comparaison avec la FIV conventionnelle.

We report here the results of a study comparing the quality of the embryos obtained after conventional IVF and after ICSI (respectively 872 and 459 embryos for 184 and 108 cycles). In the ICSI group, the female age was lower than in the IVF group, the oestradiol level on the day of hCG injection was higher, so that the number of retrieved oocytes and the number of mature oocytes. The policy of transfer being the same in the two groups, the mean number of transferred embryos was identical. The percentage of embryos without anucleate fragments, the percentage of embryos without irregularities, the percentage of 1, 2, 3, 4 or 5-cell embryos and the distribution of embryos in the 5 embryo scores were similar. In both IVF and ICSI groups, the transfer score (sum of the embryo scores of each transferred embryo) was higher for the patients who achieved pregnancy.

Limitations of the chronometric assays to determine plasma antifactor Xa activity during low molecular weight heparin therapy.

The most convenient way to estimate low molecular weight heparin (LMWH) concentration in the plasma is to determine its antifactor Xa activity. This is usually performed with specific chromogenic assays. Chronometric methods, easy to perform, have recently been introduced in clinical laboratories. This study provides evidence that these chronometric assays, are unsuitable for measuring the antifactor Xa activity in the plasma of patients receiving such heparins, because they are also sensitive to the residual antithrombin activity of LMWHs. The clearance of the antithrombin activity of a LMWH is higher than that of the antifactor Xa activity. Therefore the antifactor Xa/antifactor IIa ratio of an HBPM continuously increases after parenteral injection. It results in a significant under estimation of the antifactor Xa activity when assayed with a chronometric method because the in vitro antifactor Xa/antifactor IIa ratio of a given LMWH used to construct the calibration curve is lower than that observed ex vivo after its parenteral administration.