Enzyme Activity Assay

Theactivityoftheimmobilizedenzymeoncottonismeasuredwithporcine hemoglobin and N-benzoyl-l-arginine-p-nitroanilide (BAPNA) as substrates. The specific reactions are given schematically in Fig. 4. In the case of pepsin in solution, the activity was assayed using a solution of 2% hemoglobin in 10 mM HCl as a substrate. A volume of 1 ml enzyme extract in HCl was incubated at 37 °C with 5 ml of substrate for 10 min. The reaction was terminated using 10 ml of 5% trichloroacetic acid (TCA) and left to incubate for 5 min. The mixture was then filtered. Absorbance was recorded at 280 nm. For blank reading, TCA was added to substrate prior

Adsorption Immobilized
Fig. 4. Measurement of the activity of immobilized enzyme of cotton using N-benzoyl-L-arginine-p-nitroanilide (BAPNA) as a substrate

to the addition of enzyme extract. Specific activity (U) is expressed as:

, , Vi n x 1000x dilution factor y -absorbance value (blank) J

10 mn x mg protein in the assay

Trypsin activity in solution was assayed using BAPNA as substrate. A 43.5-mg sample of BAPNA was dissolved in 2 ml of dimethylsulfoxide, and the mixture increased to 100 ml with 0.05 M Tris-HCl buffer. A volume of 0.25 ml enzyme extract was mixed with 4 ml of freshly prepared BAPNA substrate solution and left for 10 min at 25 °C before adding 5 ml of 30% acetic acid to stop the reaction. The absorbance of the resulting solution was determined at 410 nm, where p-nitroaniline absorbs, followed by the calculation of Trypsin activity using the following formula:

absorbance value ofproduct x 1000 x volume of reaction mixture 10 mn x 8,800 x mgprotein in the assay

where 8,800 is the extinction coefficient of p-nitroaniline at 410 nm (M-1/cm).

In the case of pepsin and trypsin immobilized on the cotton, the substrates were circulated continuously into an experimental setup containing the enzyme fixed on the cotton, as described in Fig. 5. After a reaction time of 10 min, the solution was collected and analyzed as before. TCA and acetic acid were not added to the cotton to avoid denaturation of the enzyme attached to the cotton fibers.

Absorbances at 280 nm and 410 nm of the products solutions were measured with a UV-visible spectrophotometer. The amount of the enzyme

Measurments Enzyme Activity
Fig. 5. Experimental setup for measurement of the activity of immobilized enzymes

coupled onto the tosylated cotton was determined from the initial amount of protein present in the enzyme-coupling solution, subtracting the final total protein amounts present in the remaining coupling and washing solutions. The coupling yield (%) of the enzyme was then calculated from the amount of enzyme coupled to the cotton cloth divided by the initial total amount of the enzyme present in the coupling solution.

After calculating the enzyme activity, we can convert it to a concentration of transformed products, given that one unit catalyzes the hydrolysis of 1 ^mol of substrate per minute at a definite temperature and pH.

Fourier transform infrared spectra for the cotton before and after contact with tosyl chloride are shown in Fig. 6. These spectra showed that specific peaks related to -C-O-SO2-C- were observed at 1,180 and 1350/cm on the spectrum of the tosylated cotton. This indicates that tosyl chloride reacts with the primary hydroxyl groups of the cotton according to the mechanism described in Fig. 3.

On the other hand, the enzyme solution during the course of enzyme coupling to tosylated cotton was scanned in the UV-visible range from 200 to 700 nm. The spectra taken before contact with the tosylated cotton, and after 2 and 6 h of contact are shown in Fig. 7. As can be seen in this figure, the enzyme peak (at 280 nm) was higher for the solution that initially contained more enzymes. However, at 2 and 6 h, a big part of enzyme disappeared and an increase in tosyl content in the solution was observed, as expressed by an increase of the absorbance of the specific peak at 261 nm. The simultaneous increase in tosyl and decrease in enzyme concentrations clearly suggests that the degree of enzyme coupling is accompanied by a displacement of tosyl, following the nucleophilic substitution mechanism for enzyme immobilization. Therefore, it could be concluded that tosyl was covalently bound to the hydroxyl groups and behaved as a leaving agent

2000 1000 Wavenumber (cm1) Fig. 6. Fourier transform infrared spectra of pure cotton and tosylated cotton

Protein Adsorption

200 250 300 350

Wa veil umber (nm)

Fig. 7. Scanning ultraviolet spectra of the enzyme-coupling solutions taken before contact with the cotton (black squares) and at 2 (red circles) and 6 h (green triangles) after the coupling reaction

200 250 300 350

Wa veil umber (nm)

Fig. 7. Scanning ultraviolet spectra of the enzyme-coupling solutions taken before contact with the cotton (black squares) and at 2 (red circles) and 6 h (green triangles) after the coupling reaction

Fig. 8. Scanning electron micrographs of the cotton fibers before (A) and after (B) contact with the tosyl chloride solution

and was subsequently replaced by the enzyme, leading to a covalent linkage according to the mechanism shown in Fig. 3.

Scanning electron micrographs were recorded to compare the morphology of the modified cotton with that of the unmodified cotton. All samples were dried in vacuum and gold-coated. Figure 8A,B show the top surfaces of both cotton samples. We could not see any difference in the morphology of both materials, which demonstrates that no change occurred in the physical structure of the cotton fibers following activation of the hydroxyl radicals via tosylation.

Pepsin had the highest activity at pH 1.5, which decreased with increasing pH. Over pH 3 almost 90% of the relative activity was lost, and no activity was detected beyond pH 5. The optimum pH for trypsin was observed at around pH 8. No trypsin activity was measured at a pH below 4 or over 12. These values are in accordance with the data reported in literature (Devi et al. 1990). For both enzymes this process (loss of activity by changing the pH) was irreversible. The instability of acidic proteases towards the alkaline pH region contrasted with the behavior of gastric protease of many of the lower vertebrate species.

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  • david
    Can TCA stop enzyme activity?
    2 years ago
  • DUDLEY
    Why trypsin absorb 410 spectrophotometer?
    2 years ago
  • MAXIMILIAN
    How can the enzyme activity of pepsin be assessed with the BAPNA assay?
    2 months ago

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