The main objective of our laboratory is to develop a more detailed understanding of structure and function of firefly luciferase, to determine the role of the luciferase protein in the effective transformation of chemical energy into light. The investigation is based on the use of the genetic engineering and site-specific mutagenesis methods, physico-chemical methods, including enzyme kinetics and fluorescence spectroscopy. Structural studies are aimed to establish three-dimential structure of luciferase and its active center. The methods of genetic engineering will be applied to determine the role of individual amino-acid residues in substrate binding, catalysis and light production. Computer analysis was performed to reveal i) amino acid resudies involved to ATP-binding, conserved motifs in adenylating proteins; ii) amino acids responsible for bioluminescence colour. A mechanism and kinetics of regulation of luciferase activity with substrates, products and with their analogues will be examined. The role of microenvironment in luciferase catalysis will be studied measuring the enzyme kinetics and fluorescence spectra in the presence of surfactants, micelles, liposoms, nonwater solvents and other additives that mimics the enzyme surrounding in the cell.
MICROLUM IMMOLUM
Analytical concentration range, 0.01 - 100 nmol/L 0.1 - 1000 nmol/L
Storage stability 1 year at -20oC 1 year at +4oC
Number of assays per vial 25-100 10-20
Portable high sensitive luminometers with built-in stable light
standard were developed for these purposes
CLIMBI Ltd., Moscow 125422, Post Box 20, Tel: 7-095-976-4055/4428,
Fax: 7-095-976-7586.
Model LB-4A LB-4AI
Size (LxWxH), mm 200x160x110 50x210x110
Weight, kg 1.5 2.5
Power 220V, 50 Hz or 12V battery
Wattage 6 W
Light sensor PMT
Spectral range, nm 350-650
Light intensity calibration built-in stable calibration source with
emission in yellow-green region
built-in interface for connection with PC
The main features of the instrument are: high sensitivity, portability, low power, low cost and high stability. Interface was constructed which allows to transfer signal from luminometer to IBM-compatible PC. Developed software has several options for analyzing the signal: peak of intensity mode, integral signal for different integration times (1-600 s) and rate constants for increasing and decreasing of luminescence intensity. Moreover there is whole time-course of luminescence on the display for visual analysis of results. Dead time for the system is 0.1 s which much less then real time of reagents mixing.