Urease -- Characteristics and determination scheme of Worthington jack bean urease

Urease catalyzed hydrolysis of urea ：

（NH2）2CO+3H2O→CO2+2NH4OH

Jespersen (1975)  It is reported that ammonium carbamate is formed in citrate and  Tris  Produced in buffer .

Urease is found in many bacteria 、 In several yeasts and many higher plants .Varner (1960)  It was reviewed . The two best sources are ： jackbean （Canavlia ensiformis）, It has been crystallized and thoroughly studied , And Pasteurella .

This enzyme is very important in urea determination . See  Guilbault  and  Montalvo (1970). Its immobilization has been reported ： etc. .(1974)、James  and  Pring (1975)、Messing (1974)、Nakamoto wait forsomeone .(1975)、Sundaram (1973)  and  Tran-Minh  and  Broun (1975).

 

 

AI Meijie Worthington Characteristics of Jack bean urease ：

Molecular weight ：480,000 (Fishbein et al . 1970).

form ：  Monomers （a） Urease can be polymerized to form a six unit polymer of about threemillion daltons .（Fishbein wait forsomeone 1970;Fishbein  and  Nagarajan 1972a）.Andrews  and  Reithel (1970)  Sulfhydryl groups are reported .Contaxis  and  Reithel (1971)  It shows that the molecule can be divided into two parts without losing its activity . See also  Contaxis  and  Reithel (1972)、Fishbein  and  Nagarajan (1972b)、Lynn (1970)  as well as  Bailey  and  Boulter (1969).

The best  pH  value ：7.4（Cesareo  and  Langton,1992）.

Km：Tris·HCl  in  1.3mM（Cesareo  and  Langton,1992）.

Inhibitors ： Heavy metals . formation NH 4+ ion . See also  Fishbein  and  Carbone (1965). Sodium and potassium ions are inhibitors （Cesareo  and  Langton,1992）.

Specificity ： Urease has specificity for urea and hydroxyurea （Fishbein  and  Carbone 1965）. See also  Sundaram  and  Laidler (1970).

Stabilizers ： The concentration is  1 X 10 -3 M  Of  EDTA.50%  Glycerin solution can be used in  4°C  Protect urease crystal suspension for several months .

AI Meijie Worthington urease Determination method ：

Method ：Worthington  A determination method is adopted , The hydrolysis of urea is measured by coupling ammonia production with glutamate dehydrogenase reaction .

Under specified conditions , stay  25°C  and  pH 7.6  Under the condition of , One unit causes oxidation per minute  1  Micromore  NADH. In addition to improving sensitivity , The determination method also has the advantage that it can be operated to allow the quantification of urea .

Reagents ：

0.1 M  Potassium phosphate buffer ,pH 7.6

In phosphate buffer  0.023 M Adenosine-5'-diphosphate (ADP)

In phosphate buffer  0.0072 M NADH

In phosphate buffer  0.026 M a- Ketoglutarate

In phosphate buffer  1.8 M  urea

Glutamate dehydrogenase ： stay  50%  Dilute to about... In glycerol or phosphate buffer  500  Company /ml. Refrigerate during use .

enzyme ：

Apply the enzyme to  1 mg/ml  Dissolve in  0.1 M  In phosphate buffer ,pH 7.6. Before use , Further dilute in buffer to obtain  0.02-0.04 ΔA/ The rate of minutes .

Program ：

Adjust the spectrophotometer to  340 nm  and  25°C. Move the pipette into each cuvette , As shown below ：

0.10 M  Phosphate buffer ,pH 7.6 2.4  ml

0.023 M ADP 0.1  ml

0.0072 M NADH 0.1  ml

0.026 M α- Ketoglutarate  0.1  ml

1.8 M  urea  0.1  ml

GLDH (500  Company / ml ) 0.1  ml

In the spectrophotometer at  25°C  Incubate  5-10  Minutes to reach temperature equilibrium and determine the blank rate （ If there is ）. Because of the trace ammonia in the reagent , Slight changes in absorbance may be observed . After the absorbance changes to zero , Join in  0.1 ml  Properly diluted enzyme . Record  A 340 Reduce 8-10  minute . Determine from the linear part of the curve ΔA 340 / minute . There may be a slight delay .