A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine.

We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1ml saline and incubated for 15min at room temperature and then for 1h at 37°C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К=0.36±0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.

Color-shifting mutations in the C-domain of L. mingrelica firefly luciferase provide new information about the domain alternation mechanism.

We identified three color-shifting mutations-Phe467Ser, Glu490Val, and Glu490Lys-in the C-domain of the wild-type recombinant L. mingrelica luciferase. These mutations had moderate effect on the specific activity and thermal stability of the enzyme but changed the pH-dependence of its bioluminescence spectra. We constructed the model structures of the enzyme in three known conformations (open, adenylation, and oxidation conformation). The structural analysis and experimental data provided no evidences that these residues participate in structure-forming interactions in the open or oxidation conformation or that their mutations alter the overall structure of the enzyme. Given that the bioluminescence spectra reflect the microenvironment of the emitter (oxyluciferin in an electronically excited state), we concluded that the mutated residues affect the active site during the emission of light via short-range interactions. We found that it is only in the adenylation conformation that the residues Phe467 and Glu490 approach the N-domain, whereas the domain rotation associated with the oxidation conformation completely removes them from the active site. Therefore, the emission most likely occurs from the adenylation conformation.

Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.

Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.