Uricase - Characteristics of uricase in Worthington pig liver:

Urate ：O 2 Oxidoreductase

Uricase （ Uric acid oxidase ） Catalyze the following overall reactions ：

Urate+O2+H2O→Allantoin+H2O2+CO2

This reaction represents the exception of human 、 Termination of purine catabolism in all mammals except higher apes and Dalmatians .

Mahler (1963)  This enzyme is reviewed .

Uricase is very important for the determination of uric acid in biological fluids . The reaction is not only specific , You can also do it in  292 nm、340 nm  Or colorimetric monitoring by coupling the color response . Non enzymatic methods will be affected by turbidity or the presence of aspirin 、 ascorbic acid 、 Glutathione 、 Paracetamol and many antibiotics interfere . See  Itiaba wait forsomeone .（1975）; Guan et al .（1975）; Pace et al .（1974）;Gökicke  and  Gökicke (1973); Kabasakalian et al .（1973）; Ram and Gambino  (1973); Steele  (1970)  And Troy and paddy  (1970).

 

 

AI Meijie  Worthington Characteristics of pig liver uricase ：

Molecular weight ：125,000 (Pitts et al . 1974).

form ： The enzyme is produced by  32,000 MW  It consists of four subunits , Each molecule  (125,000 MW)  There is a copper atom  (Pitts et al . 1974).

The best  pH  value ：9.0 (Mahler 1963).

Extinction coefficient ：= 11.3  Extinction coefficient (Mahler 1963).

Isoelectric point ：6.3 (Mahler 1963).

Inhibitors ： Various purine analogues of urate （Bergmann wait forsomeone 1963;Baum wait forsomeone 1956）、 Cyanide and other copper chelators .Fridovich (1965)  The report says , Urate in alkaline solution can be converted into effective inhibitor , Oxate .

Specificity ： The enzyme is highly specific for uric acid .（ See Mahler  1963  year ）.

stability ： stay  10%  Uricase purified in saturated ammonium sulfate is  5°C  It can be stable for a year .

AI Meijie Worthington Uricase Determination method ：

Method :  The reaction rate is caused by measuring the oxidation of uric acid to allantoin  290 nm  Determined by the decrease of absorbance at . Under certain conditions , A unit in  25°C  and  pH 8.5  Oxidize every minute  1  Micromolar uric acid .

Reagents ：

1.0.1 M  Sodium borate buffer ,pH 8.5

2.0.12 mM  uric acid . take  60 mg  Lithium carbonate is dissolved in  15 ml  Water and filter . By way of  100 mg  Uric acid is dissolved in the filtrate to prepare a fresh solution . It may need to be heated to  50-60°C  To produce a solution . Cool and dilute with reagent grade water to  100 ml. use  0.1 M  Borate dilution  1/100,pH 8.5.

Be careful ： Before using ,0.1 M  Sodium borate buffer and  0.12 mM  Uric acid solution should be prepared by adding  O 2 Bubbling through solution  10-15  Minutes for oxidation . Every time  20  Oxygenate again in minutes .

enzyme ：

With  1 mg/ml  Dissolve in cold  (5°C) 0.1 M  Sodium borate buffer ,pH 8.5.

equation ：

Just before the determination , Further dilute to  0.01-0.1  Company /ml  The concentration of .

Program ：

Adjust the spectrophotometer to  290 nm  and  25°C.

The pipette is as follows ：

Borate buffer  0.5  ml

0.12  Mm uric acid  2.0 ml

Incubate in a spectrophotometer  4-5  Minutes to reach temperature equilibrium and determine the blank rate （ If there is ）. Add at zero time  0.5 ml  Enzyme and record  A 290 The fall of 6-7  minute . Calculate from the initial linear part of the curve ΔA 290 .

Worthington Core enzyme ： Include ： Collagenase , Deoxyribonuclease I, Elastase , Papain , Ribonuclease , Trypsin, etc .