Ureasa de Canavalia ensiformis (judía Jack), CAS No.9002-13-5

CAS: 9002-13-5 Cat. No.: U128713 Número EC: 232-656-0

GRADE & PURITY EnzymoPure™, ≥45 units/mg dry weight

Descripción general

Jespersen (1975) informa de que el carbamato de amonio se produce en tampón citrato y Tris.
La ureasa se encuentra en muchas bacterias, en varias especies de levaduras y en varias plantas superiores. Varner (1960) la ha revisado. Dos de las mejores fuentes son: Los frijoles Jack (Canavlia ensiformis), de los que se ha cristalizado y estudiado a fondo, y el Bacillus pasteurii.
La enzima es importante en la determinación de la urea. Véase Guilbault y Montalvo (1970). Su inmovilización ha sido reportada: et al. (1974), James y Pring (1975), Messing (1974), Nakamoto et al. (1975), Sundaram (1973) y Tran-Minh y Broun (1975).
Características de la Ureasa de Jack Bean:
Km: 1,3mM en Tris⋅HCl (Cesareo y Langton, 1992).
Especificidad
La ureasa es específica para la urea y la hidroxiurea (Fishbein y Carbone 1965). Véase también Sundaram y Laidler (1970).
Composición
La (a)ureasa monomérica puede polimerizarse para formar polímeros de seis unidades de unos tres millones de daltons. (Fishbein et al. 1970; Fishbein y Nagarajan 1972a). Andrews y Reithel (1970) informan sobre los grupos sulfhidrilo. Contaxis y Reithel (1971) indican que la molécula puede dividirse por la mitad sin pérdida de actividad. Véase también Contaxis y Reithel (1972), Fishbein y Nagarajan (1972b), Lynn (1970), y Bailey y Boulter (1969).
PH óptimo：7,4
Estabilizadores
EDTA en concentraciones de 1 X 10-3 M. Las soluciones de glicerol al 50% protegen la suspensión cristalina de ureasa durante varios meses a 4°C.
Inhibidores
Metales pesados. Formación de iones NH4+. Véase también Fishbein y Carbone (1965). Los iones sodio y potasio son inhibidores (Cesareo y Langton, 1992).
Ensayo de la ureasa
Método
Worthington ha adoptado un método de ensayo en el que la hidrólisis de la urea se mide acoplando la producción de amoníaco a una reacción de glutamato deshidrogenasa.
fórmulas
Una unidad resulta en la oxidación de un micromol de NADH por minuto a 25°C y pH 7.6 bajo las condiciones especificadas. Además de una mayor sensibilidad, el método de ensayo posee la ventaja de que puede manipularse para permitir la cuantificación de la urea.
Reactivos
0.tampón fosfato potásico 1 M, pH 7,6
0.023 M Adenosina-5'-difosfato (ADP) en tampón fosfato
0.0072 M NADH en tampón fosfato
0.026 M a-Cetoglutarato en tampón fosfato
1.8 M Urea en tampón fosfato
Glutamato deshidrogenasa: Diluir a aproximadamente 500 unidades/ml en glicerol al 50% o tampón fosfato. Conservar en frío durante su uso.
Enzima
Disolver la enzima a un mg/ml en tampón fosfato 0,1 M, pH 7,6. Inmediatamente antes del uso, diluir más en tampón para obtener una velocidad de 0,02-0,04 ΔA/minuto.
Procedimiento
Ajustar el espectrofotómetro a 340 nm y 25°C. Pipetear en cada cubeta de la siguiente manera:

0.tampón fosfato 10 M, pH 7,6 2,4 ml

0.023 M ADP 0,1 ml

0.0072 M NADH 0,1 ml

0.026 M α-Cetoglutarato 0,1 ml

1.8 M Urea 0,1 ml

GLDH (500 unidades/ml) 0,1 ml

Incubar en el espectrofotómetro a 25°C durante 5-10 minutos para lograr el equilibrio de temperatura y establecer la tasa de blanco, si la hubiera. Puede observarse un ligero cambio en la absorbancia debido a trazas de amoníaco en los reactivos. Una vez obtenido un cambio cero en la absorbancia, añadir 0,1 ml de enzima diluida adecuadamente. Registrar la disminución de A340 durante 8-10 minutos. Determinar ΔA340/minuto a partir de la porción lineal de la curva. Puede producirse un ligero desfase.

Cálculo

Specifications

Product Name

Ureasa de Canavalia ensiformis (judía Jack), CAS No.9002-13-5

Sinónimos

Urea amidohydrolase

Grado

EnzymoPure™

Especificaciones y pureza

EnzymoPure™, ≥45 unidades/mg de peso seco

Mecanismos bioquímicos y fisiológicos

La ureasa cataliza la hidrólisis de la urea en dióxido de carbono y amoníaco. La ureasa interviene en el metabolismo del nitrógeno y la degradación de la urea. La ureasa de Canavalia ensiformis une 2 iones de níquel por subunidad.

CAS

9002-13-5

Número de la Comisión de Enzimas

3.5.1.5

Tipo de molécula

Enzyme

Almacenamiento y envío

Concentración

≥45 units/mg dry weight

Condiciones de almacenamiento de almacenamiento

Conservar a -20°C

Enviado en

Hielera + almohadillas de hielo

Definición de unidad

One Unit oxidizes one micromole of NADH per minute at 25°C, pH 7.6. The hydrolysis of urea is measured by coupling ammonia production to a glutamate dehydrogenase reaction.

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