Bioluminescence: Principles and Applications

of CHEMICAL ENZYMOLOGY DIVISION

The main research items:
STRUCTURAL AND FUNCTIONAL INVESTIGATIONS OF FIREFLY LUCIFERASE

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.

BIOLUMINESCENT ATP-ASSAY
Bioluminescent analysis has already proved to provide wide opportunities in the area of ecology, medicine, hygiene and food control. It is important to have results of analysis in real time just in the place where samples were picked up. Our laboratory is the well-known research center on Bioluminescence and on development of methods and reagents for bioluminescent microassay. Bioluminescent ATP-metry has become the basis for the so called юRapid Microbiologyю. Several applications were proposed for:
MEDICINE: 3-hour test for antibiotic susceptibility and MIC; 5 - hour test for bacterial contamination in wounds and tissues, Bioluminescent test for monitoring of Blast Transformation Reaction (instead of radioactive test)
ECOLOGY: Bioluminescent tests for estimation of bacterial contamination of natural and drinking water; bioluminescent test for active sludge control during waste water purification
TECHNOLOGY AND BIOTECHNOLOGY: Express test for bioresistance against fungi's action and biocide activity control for polymers, lubricants, fuel,etc.; 5-minutes test for biocatalyst activity control.
FOOD QUALITY CONTROL: Express method for bacterial contamination assessment in raw milk, milk products, meat, juices, etc. Bioluminescent ATP-Reagent Kits are provided with CLIMBILUM Minilab. All kits include Bioluminescent ATP-Reagent and ATP standard solution. Bioluminescent ATP-Reagents are lyophilized ready-to-use mixtures based on the soluble (MICROLUM) or immobilized (IMMOLUM) firefly luciferase, luciferin, buffer salts and stabilizers.

Basic properties of the BL-ATP-Reagents
                                  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.

Technical parameters of CLIMBI luminometrs
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.

For further information please contact:
Chemical Enzymology division, Chemistry Department,
Lomonosov Moscow State University, Moscow 119899, Russia
Tel. 7(095)939-2660,
Fax: 7(095)939-3589,
E-mail: UNN@enzyme.chem.msu.su
Researches:
Webmaster@www.chem.msu.su