RESUMO
In our system, urease/AlaDH have been encapsulated within erythrocytes by using slow dialysis methods. Urea is decomposed into ammonia and bicarbonate and the ammonia released is converted into alanine by reacting pyruvate under the catalytic action of AlaDH. It is very important for our that products are formed quickly but the ammonia is not connected definetely. For this aim, urease/AlaDH we encapsulated using different enzyme activity ratio (0.5:1.5; 0.5:2.5; 0.25:1.25 U/U urease/AlaDH). The activities of enzyme systems, encapsulation yield, McV, McH, and McHc were measured for each sample. Investigated results suggest that loaded enzyme systems can be used as potential carrier systems for the removal of high levels of urea from blood.
Assuntos
Aminoácido Oxirredutases/farmacologia , Eritrócitos/enzimologia , Ureia/metabolismo , Urease/farmacologia , Alanina Desidrogenase , Aminoácido Oxirredutases/metabolismo , Amônia/metabolismo , Cápsulas , Humanos , Técnicas In Vitro , Ácido Pirúvico/metabolismo , Urease/metabolismoRESUMO
Reactive partially reduced oxygen species such as superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are produced in aerobically growing organisms during normal cellular respiration. To provide an effective defense against these reactive species, many aerobic organisms have evolved a multienzyme defense which includes superoxide dismutase, catalase and peroxidase. The superoxide anion may cause appreciable cellular damage by oxidizing aminoacids or by causing DNA strand breakage. Catalase was covalently immobilized on activated methoxypolyethyleneglycol-5000 and catalase and PEG-catalase were encapsulated in erythrocyte. Enzyme activity, encapsulation yield and hemograme analysis were determined for each sample. The erythrocyte shape of the samples were investigated by using phase contrast microscopy.
Assuntos
Catalase/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Eritrócitos/enzimologia , Polietilenoglicóis/farmacocinética , Catalase/química , Catalase/metabolismo , Composição de Medicamentos/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacocinética , Eritrócitos/citologia , Eritrócitos/metabolismo , Testes Hematológicos , Humanos , Microscopia de Contraste de Fase , Polietilenoglicóis/química , Polietilenoglicóis/metabolismoRESUMO
No intravenously injectable enzyme preparate containing urease as an alternetive to hemodialysis, hemoperfusion and CAPD systems in patients having chronic renal failure has been encountered in literature. In this study, it has been aimed to convert blood urea to alanine by using PEG-urease/PEG-AlaDH enzyme pair encapsulated within living erythrocyte. In this system, urea is decomposed into NH3 and HCO3- and the ammonia released is converted into alanine by reacting pyruvate under the catalytic action of alaninedehydrogenase. The production of pyruvate and NADH by erythrocyte required in the second stage of the reaction will make the process a feasible and ceaseless one. The success of the system will enable the renal patients with diabetes mellitus. Urease and AlaDH were covalently immobilized on activated PEG. PEG-urease/PEG-AlaDH were encapsulated in erythrocyte (1/1)(v/v) by using slow dialysis methods. The activity of enzyme system, encapsulation yield and hemogram analysis were determined for each sample.
Assuntos
Aminoácido Oxirredutases/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Eritrócitos/enzimologia , Urease/farmacocinética , Alanina/biossíntese , Alanina Desidrogenase , Aminoácido Oxirredutases/sangue , Aminoácido Oxirredutases/metabolismo , Amônia/análise , Diabetes Mellitus/terapia , Composição de Medicamentos , Sistemas de Liberação de Medicamentos/normas , Enzimas Imobilizadas/sangue , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacocinética , Testes Hematológicos , Humanos , Cinética , Polietilenoglicóis , Ácido Pirúvico/análise , Urease/sangue , Urease/metabolismoRESUMO
Erythrocytes can be used to entrap drugs, enzymes or other molecules with active properties, with various encapsulation procedures. The carrier is nonimmunogenic, biodegradable, and circulates freely throughout the body. Urease was covalently immobilized on activated methoxypolyethyleneglycol-5000 (PEG-5000) (1:3 molar ratio). Urease and PEG-Urease were encapsulated in erythrocyte (1/1) (v/v) by using slow dialysis methods. To optimize the loading of erythrocyte, the above base procedure was varied to test the effect of some parameters. Dialysis time, dialysis temperature, storage condition for erythrocyte conjugate, Urease and PEG-Urease concentration were investigated.
Assuntos
Sistemas de Liberação de Medicamentos , Eritrócitos/metabolismo , Polietilenoglicóis/metabolismo , Urease/metabolismo , Tamanho Celular , Diálise/normas , Composição de Medicamentos/métodos , Composição de Medicamentos/normas , Enzimas Imobilizadas , Humanos , Temperatura , Fatores de TempoRESUMO
Urease (E.C 3.5.1.5) was covalently immobilized on activated methoxypolyethyleneglycol-5000 which is linear, uncharged, soluble in water and nonimmunogenic. mPEG is bound to the epsilon-NH2 groups of Lysin in urease. Previously different molar ratios of urease -Lys/activated-mPEG were searched for immobilization. Storage stabilities, molecular weights and the values of blocked amino groups were determined for each immobilized urease and the best conditions was found 1:3 urease-Lys/activated mPEG. Furthermore physical characterization, kinetic constants (Km, Vmax), heat and temperature stabilites were also determined.