2.4. HIV-1 Protease Activity Assay

HIV-1 protease activity was determined according to the method of AnaSpec, Fremont, California, USA, utilizing the SensoLyte® 520 HIV-1 protease fluorimetric assay kit (Cat no. AS-71147, AnaSpec, Fremont, California, USA) and black, flat-bottom 96-well microplates with nonbinding surfaces (Cat no. CLS3600, Merck, Darmstadt, Germany). Assay working solutions were freshly prepared by following the instructions with regards to the enzyme assay buffer, active recombinant HIV-1 protease stock solution (0.2 mg/ml; Cat no. 72028-5, AnaSpec, Fremont, California, USA), HIV-1 fluorescence resonance energy transfer (FRET) substrate, and the reference aspartic acid protease (pepstatin A) inhibitor.

In order to determine the optimal enzyme activity response, the following HIV-1 protease combinations were evaluated: (i) 800-fold diluted HIV-1 protease stock in 1x enzyme buffer (40 μl) and nuclease-free water (10 μl), (ii) 80-fold diluted HIV-1 protease stock in 1x enzyme buffer (40 μl) and nuclease-free water (10 μl), and (iii) to evaluate the effect of the inhibitor, water was replaced with pepstatin A solution (10 μl) in each reaction combination.

The enzymatic reactions were initiated by adding HIV-1 protease FRET substrate solution (50 μl) and plates incubated at 25°C for 15 min. Reactions were performed in triplicate. The fluorescence intensity of the HIV-1 protease activity was measured at an excitation (Ex) wavelength of 490 nm and emission (Em) wavelength of 520 nm in minute intervals for 60 min using a fluorescence kinetic synergy MX analytical diagnostic reader (Gen spectrophotometer, Bio Tek, Vermont, USA). Data were transferred to a Microsoft Excel spreadsheet for further analysis. The average data generated were plotted against time.

The following reaction combinations were prepared: (i) 80-fold diluted HIV-1 protease stock in 1x enzyme buffer (40 μl) and plant extract (10 μl) from each concentration (16, 1.6, 0.16, and 0.016 mg/ml), respectively, (ii) positive controls containing 80-fold diluted HIV-1 protease stock in 1x enzyme buffer (40 μl) and nuclease-free water (10 μl), and (iii) inhibitor controls containing 80-fold diluted HIV-1 protease stock in 1x enzyme buffer (40 μl) and pepstatin A (10 μl). The enzymatic reactions were initiated by adding HIV-1 protease FRET peptide substrate solution (50 μl) and plates incubated at 25°C for 15 min. The reactions were performed in triplicate. Microplates were processed as described above. The percentage inhibition of each extract activity was calculated as

where Abs = absorbance. Graphs were generated by plotting inhibition percentage values against extract concentrations. The half maximal inhibitory concentration (IC₅₀) was calculated from the percentage values.