A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING:
IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING
CAPILLARY ELECTROPHORESIS

a 2014

A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS

SCHEJBAL, Jan, Roman ŘEMÍNEK, Lukáš ZEMAN, Marta PELCOVÁ, Zdeněk GLATZ et. al.

Základní údaje

Originální název

A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS

Název česky

Nový přístup při screeningu inhibitorů cytochromů P450: Imobilizovaný enzymatický reactor pro on-line studie využívající kapilární elektroforézu

Autoři

SCHEJBAL, Jan, Roman ŘEMÍNEK, Lukáš ZEMAN, Marta PELCOVÁ a Zdeněk GLATZ

Vydání

Book of abstracts, CEITEC Annual Conference 2014 "Frontiers in material and life sciences" 2014

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10600 1.6 Biological sciences

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Organizační jednotka

Středoevropský technologický institut

Klíčová slova česky

Capillary electrophoresis, Cytochrome P450 2C9, IMER, Diclofenac, Sulfaphenazole, Kineticts, Inhbition

Klíčová slova anglicky

kapilární elektroforéza, cytochrom P450 2C9, IMER, diclofenac, sulfafenazol, kinetika, inhibice

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 26. 11. 2014 15:55, Mgr. Jan Schejbal, Ph.D.

Anotace

V originále

In this work we present an IMER integrated into capillary based on CYP 2C9 isoform, representing approximately 20 % of all CYP in human liver which is responsible for metabolizing more than 10 % of commonly used drugs. The CYP 2C9 was immobilized on magnetic microparticles Si-MAG carboxyl using the carbodiimide method and the formation of IMER was established by two permanent magnets placed in cassette for capillary electrophoresis. The enzymatic reaction was instantly followed by capillary zone electrophoresis separation so that the product could be detected and quantified with UV-DAD. Unfortunately CYP enzymes are membrane bound, which makes their immobilization rather cumbersome and displayed features are usually inferior in comparison to soluble enzymes, further optimization of incubation and separation was therefore necessary. All challenges were in the end overcome and optimized method was used to determine Michalis Menten constant for diclofenac and to characterise the inhibition of previous reaction by sulphophenazole. All acquired data were in good correlation with literature, proving this method suitable for inhibition studies. Furthermore the consumption of enzyme and reactants is significantly lower compared to generally used methods like fluorescence microassays.

Návaznosti

GAP206/10/0057, projekt VaV

Název: Miniaturizovaný systém pro "on-line" studie metabolismu léčiv založený na kapilární elektroforéze

Investor: Grantová agentura ČR, Miniaturizovaný systém pro "on-line" studie metabolismu léčiv založený na kapilární elektroforéze

Citovat

SCHEJBAL, Jan, Roman ŘEMÍNEK, Lukáš ZEMAN, Marta PELCOVÁ a Zdeněk GLATZ. A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS. In Book of abstracts, CEITEC Annual Conference 2014 "Frontiers in material and life sciences". 2014.

@proceedings{1210569,
   author = {Schejbal, Jan and Řemínek, Roman and Zeman, Lukáš and Pelcová, Marta and Glatz, Zdeněk},
   booktitle = {Book of abstracts, CEITEC Annual Conference 2014 "Frontiers in material and life sciences"},
   keywords = {kapilární elektroforéza, cytochrom P450 2C9, IMER, diclofenac, sulfafenazol, kinetika, inhibice},
   language = {eng},
   title = {A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS},
   year = {2014}
}

TY  - CONF
ID  - 1210569
AU  - Schejbal, Jan - Řemínek, Roman - Zeman, Lukáš - Pelcová, Marta - Glatz, Zdeněk
PY  - 2014
TI  - A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS
KW  - kapilární elektroforéza, cytochrom P450 2C9, IMER, diclofenac, sulfafenazol, kinetika, inhibice
N2  - In this work we present an IMER integrated into capillary based on CYP 2C9 isoform, representing approximately 20 % of all CYP in human liver which is responsible for metabolizing more than 10 % of commonly used drugs. The CYP 2C9 was immobilized on magnetic microparticles Si-MAG carboxyl using the carbodiimide method and the formation of IMER was established by two permanent magnets placed in cassette for capillary electrophoresis. The enzymatic reaction was instantly followed by capillary zone electrophoresis separation so that the product could be detected and quantified with UV-DAD. Unfortunately CYP enzymes are membrane bound, which makes their immobilization rather cumbersome and displayed features are usually inferior in comparison to soluble enzymes, further optimization of incubation and separation was therefore necessary. All challenges were in the end overcome and optimized method was used to determine Michalis Menten constant for diclofenac and to characterise the inhibition of previous reaction by sulphophenazole. All acquired data were in good correlation with literature, proving this method suitable for inhibition studies. Furthermore the consumption of enzyme and reactants is significantly lower compared to generally used methods like fluorescence microassays.
ER  -

SCHEJBAL, Jan, Roman ŘEMÍNEK, Lukáš ZEMAN, Marta PELCOVÁ a Zdeněk GLATZ. A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS. In \textit{Book of abstracts, CEITEC Annual Conference 2014 ''Frontiers in material and life sciences''}. 2014.

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