Enzymatic Activity Measurements

Urease catalyzes the hydrolysis of urea, producing ammonium and carbonate ions according to the following reaction:

Determination of the activities of the free and immobilized urease is carried out by measuring the production of ammonium according to a procedure given by Sigma (Sigma catalog no. 535) with a blood urea nitrogen (BUN) acid reagent and BUN color reagent (Sigma). A calibration curve is established by mixing 3 ml of BUN acid reagent with 2 ml of BUN color reagent and 1 ml of sample solution (urea concentration between 2 and 60 mM) and maintaining the mixture at precisely 80 °C for 10min. After cooling, the respective absorbances at 524 nm are recorded (Coche-Guerente et al. 1995).

Uricase activity is measured by spectrophotometric detection of the uric acid according to a Sigma procedure (Sigma catalog no. 292). The reaction is monitored by the absorbance change at 292 nm. Since uric acid absorbs at 292 nm and the end product of the reaction does not, the decrease in absorbance is proportional to the decrease in the uric acid concentration and thus to the uricase activity (the extinction coefficient of the uric acid solution at 292 nm, e292 = 12,200/mol/cm).

solution of freeureaseor biotinylated urease biotinylated ureasesolution after textile immersion

rinsed solution urea measurement

Initial enzymeactiviy (U.mg-1)

Transformation rate (U)

weight of enzyme on the textile initial enzyme weight - enzyme weight after textile immersion - enzyme wieght in rinsed solution

Textile specific activity= textile activity / weight of enzyme

Fig. 11. Description of the procedure used for the determination of the specific enzyme activity of the textile

Xanthine oxidase activity is measured by spectrophotometric detection at 292 nm of the produced uric acid or from the amount of hydrogen peroxide produced with xanthine as substrate (Coche-Guerente et al. 1995). The hydrogen peroxide concentration is determined according to analytical methods that have been described previously (Green and Hill 1984; peroxidase activity determination from SIGMA) based on enzymatic reactions with peroxidases.

The enzyme-specific activity of the modified textiles is calculated in the case of urease immobilization after determining the weight of the enzyme on the textile and the enzyme activity of the material. Figure 11 depicts the measuring procedure used to estimate the weight of enzyme immobilized on thetextile. Therecordingoftheactivity oftheenzymesolutionallowsus to know the corresponding weight of enzyme (knowing the initial specific activity of the enzyme).

Ion-exchange textiles have been used to achieve enzyme immobilization, as their ion-exchanging groups can react with the biotin groups of biotinylated enzymes. Three types of textile have been used: carboxylic acid, tertiary amine and quaternary ammonium groups, as cation and anion exchangers, respectively (Fig. 12).

Table 4 shows the evolution of the ion exchange capacity (EC) of a textile before and after chemical modification (biotin attachment). In the case of the carboxylic acid tissue, the EC value is in good agreement with the proposed reaction scheme for the attachment ofbiotin to the COO- groups.

Fig. 12. Chemical reactions used to modify a textile support by biotin attachment: (A) cation exchanger textile (carboxylic acid groups) and (B) anion exchanger textile bearing amino groups
Table4. Exchange capacity of different textiles before and after chemical modification (biotin anchorage). Carbox Carboxylic acid, N Tert tertiary amine, N Quat quaternary ammonium, EC exchange capacity, Max maximum

Carbox Carbox Biotin

N Tert N Tert Biotin

N Quat

N Quat Biotin

EC (meq/g)a

3.0 2.3

1.8 2.6

0.3

2.2

Exchange

2-5 -

Max -

0.4-1

-

capacity (meq/g)b

1.6

"Measured in this work bFrom IFTH data

"Measured in this work bFrom IFTH data

After the reaction, the textile lost its ion EC. Conversely, in the case of tertiary amine groups, these groups are quaternized during the reaction and the anion EC is increased after modification.

Was this article helpful?

0 0

Post a comment