Xanthine Oxidase and Uricase Immobilization

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The efficiency of the immobilization method being demonstrated with urease and in order to prove the potentiality of the molecular recognition

Xanthine Oxidase
Fig. 15. Catalytic chemical reactions achieved by xanthine oxidase and uricase

process, different types of enzyme have been fixed onto textiles: xanthine oxidase, which allows the chemical transformation of xanthine into uric acid, and uricase, which transforms uric acid into allantoin (see the reaction scheme shown in Fig. 15). The enzymatic transformation of xanthine into uric acid determined from the increase in the absorbance at 290 nm (production of uric acid) is shown in Fig. 16, for the different studied ion exchanging textiles.

0,12

0,04

o in

time (min)

Fig. 16. Absorbance values recorded at 290 nm at the exit of the column after injection of 40 ml of a xanthine (1.2x 10-4 M ) in 5x10-2 M phosphate buffer solution (pH 7.5)

time (min)

Fig. 16. Absorbance values recorded at 290 nm at the exit of the column after injection of 40 ml of a xanthine (1.2x 10-4 M ) in 5x10-2 M phosphate buffer solution (pH 7.5)

Fig. 17. Experimental device for a sequential bienzymatic process

After having demonstrated the feasibility of this method for xanthine substrate removal, we have sophisticated the process by simply adding in the column another textile modified by uricase. Figure 17 represents the set-up used in a sequential bienzymatic process, which allows both the degradation of xanthine and of uric acid, which is the reaction product of the first enzyme reaction in the flow cell.

Spectrophotometric measurements have been achieved at the exit column. Two different wavelengths have been used to demonstrate the decrease in xanthine concentration (273 and 290 nm). The production of uric acid would result in an increase in the absorbance at 290 nm; however, a reduction in the optical density recorded at 290 nm was observed, and thiswasattributedtothereductioninxanthineconcentration, which would lead to an increase, if the second reaction involving uricase was not effective . In fact this means that no production of uric acid can be recorded, as the reaction across the second enzyme layer immobilizing uricase is highly efficient. The observed decrease in fact corresponds to the reduction in xanthine concentration. Even if the main optical absorption region of xanthine is 273 nm, it also absorbs at 290 nm (main absorption band of uric acid), but with a lower absorption coefficient. At these two wavelengths,

Xanthine Absorbance

Fig. 18. Absorbance change versus time measured at 273 (pink squares) and 290 nm (navy blue diamonds) in the flow at the exit of the column

Fig. 18. Absorbance change versus time measured at 273 (pink squares) and 290 nm (navy blue diamonds) in the flow at the exit of the column the slopes of the two linear variations of absorbance with time are shown in Fig. 18, allow us to calculate the reaction rate decrease knowing the molar extinction coefficients at 273 and 290 nm, respectively. The same obtained value of the reaction rate (12 ^mol/min) demonstrates that no production of uric acid occurred during the time of the experiment, during which all of the xanthine was converted to allantoin. The uric acid enzymatically produced in the course of the flow across the first textile is totally consumed by the enzymatic reaction on the second textile. These data show the high efficiency of the bienzymatic process, which results in the sequential action of the two enzyme modified textiles.

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