反应详情

Substrate stock solutions were prepared by dissolving ˜50 mg BAPNA or suc-AAPF-PNP in DMSO; these stock solutions were then diluted with DMSO and 0.1 M TRIS/HCl buffer (pH 7.4) to provide substrate solutions of various concentrations (final concentration of DMSO in each substrate solution was 15% v/v). A measured quantity of particles, 30-100 mg, were placed in a microcentrifuge tube, 1.3 ml of substrate solution was added, and the mixture was bath-sonicated to completely disperse the particles. The mixture was then placed in a water bath at 30° C. for 5-6 hours, with additional agitation provided every 45-60 minutes. The mixture was then removed from the water bath and centrifuged at 14,000 rpm for 30 minutes. The reaction time was measured from initial sonication to the beginning of centrifugation. Catalytic activity was determined from the increase in free p-nitrophenol concentration, measured by UV/VIS spectroscopy at 410 nm with an extinction coefficient of 8500 M−1 cm−1. The measured absorbance was compared to that of the substrate solution at 410 nm and from the absorbance due to light scattering of the particle dispersion of particles in 0.1 M Tris buffer (prepared identically as above) at 410 nm. These contributions to the absorbance were subtracted from the total absorbance at 410 nm to obtain the absorbance due to free p-nitrophenol. Each data point was calculated based on the average of 3 to 7 trials.

1. customSubstrate stock solutions were prepared
2. customto provide substrate
3. concentrationsolutions of various concentrations (final concentration of DMSO in each substrate solution
4. customA measured quantity of particles, 30-100 mg, were placed in a microcentrifuge tube
5. addition1.3 ml of substrate solution was added
6. customthe mixture was bath-sonicated
7. additionto completely disperse the particles
8. customprovided every 45-60 minutes
9. customThe mixture was then removed from the water bath
10. waitcentrifuged at 14,000 rpm for 30 minutes
11. customsonication to the beginning of centrifugation
12. temperatureincrease in free p-nitrophenol
13. concentrationconcentration
14. customfrom the absorbance due to light scattering of the particle dispersion of particles in 0.1 M Tris buffer (prepared identically as above) at 410 nm