Total Length: 1623 words ( 5 double-spaced pages)
Total Sources: 5
Page 1 of 5
Biochemical Analysis: Dengue Denv Protease
Dengue Virus Protein Biochemical Analysis
Database Search Methods
Of the several CSU databases available, I chose to utilize PubMed, because Medline is specific for biological research. I also wanted to avoid retrieving qualitative studies, given the topic chosen, and felt Pubmed would be the best way to find quantitative studies.
The first search string I used was
In the study being reviewed here, protease activity was measured by an increase in fluorescence activity at 440 nm after excitation at 350 nm using a fluorescent spectrophotometer plate reader (Rothan et al., 2012a). The assay design chosen by Rothan and colleagues (2012a) is called a homogenous assay because the product or substrate does not need to be isolated from the reaction mixture before quantification (Zhang, 2012). The 30 minute incubation period prior to reading fluorescent activity is also consistent with enzyme dynamics that reach equilibrium rapidly.
The main ingredient for the protease essay was a single-chain recombinant protein containing the sequence of both NS3 and NS2B (NS2B-NS3pro) (Rothan et al., 2012a). By producing a single chain polypeptide, the authors avoided the need to perform an association step to bring NS3 and NS2B together. The other main ingredients were recombinant RC-1 and the substrate Boc-GRR-AMC. A Tris-HCl buffer was prepared (pH 8.5) that acted as the reference condition, because it should have minimal or undetectable fluorescent activity at 440 nm even in the presence of substrate. The second condition was buffer plus 2 ?M NS2B-NS3pro, which should produce fluorescence activity in the presence of substrate in a concentration-dependent activity. The results of these two conditions when incubated for 30 minutes at three different temperatures are shown in Figure 1. The three temperatures represent the conditions under which DENV protease will be expected to help the virus replicate, which are 28?C for a mosquito host, 37?C for an uninfected human host, and 40?C for an infected human host with a high fever. If fluorescence is produced in a concentration-dependent manner, then this is proof the assay works as expected.
The third condition was buffer with NS2B-NS3pro (2 ?M) and substrate (20 ?M) at constant concentrations, while titrating recombinant RC-1 (Rothan et al., 2012a). This assay was also performed at the three most relevant temperatures of 28?C, 37?C, and 40?C. If the fluorescent signal generated by NS2B-NS3pro cleavage of the substrate is inhibited in a concentration-dependent manner by RC-1, then this supports the theory that the defensin RC-1 can inhibit DENV protease in vivo and by extension, dengue viral replication.
The basic approach for conducting these assays would be to allocate a fixed volume to wells within a multi-well plate that can be read in the spectrophotometer (Rothan et al., 2012a). For the first experiment, there were three temperatures and six different concentrations of substrate. Since each experiment was run in triplicate, this would require 54 wells. The second experiment was performed at three different temperatures, in triplicate, with six different concentrations of RC-1 (null condition now shown in figure 4) and would therefore need 54 wells as well. The way this protease assay was designed enabled Rothan and colleagues (2012a) to determine the substrate Km values and the RC-1 IC50 (half maximal inhibition) at all three temperatures.
Results
Figure 3 shows the enzyme kinetics of NS2B-NS3pro acting upon the Boc-GRR-AMC substrate at the three temperatures (Rothan et al., 2012a). Six different NS2B-NS3pro concentrations were used, including buffer alone, which generated three temperature-dependent saturation curves. The highest protease activity was exhibited at the temperature of mosquitoes. The next highest activity was at normal human body temperature. Much lower activity was observed at 40?C, the body temperature of an infected individual running a high fever. The results shown in Figure 3 reveal the assay works as expected and that the DENV protease would be….....