Marlene DeLuca

[1] Purification and properties of firefly luciferase

Publisher Summary This chapter discusses purification and properties of firefly luciferase. Firefly luciferase in the presence of luciferin (d-LH2), adenosine triphosphate (ATP)-Mg, and molecular oxygen catalyzes the production of light. The color of the emitted light shows a peak at around 560 nm. This emission can be affected by temperature, pH, and metal ions. At low pH or in the presence of lead, mercury, or other heavy metals, the emission peak is shifted to red, showing an emission around 615 nm. Thus, when this system is used for the assay of ATP, it is critical to run internal controls to make sure that the light color has not been shifted. Because of the greater sensitivity of phototubes in blue in contrast to the red, a shift to red will give an apparent lower level of ATP. A dark-adapted eye can readily discern the difference in color between the control and one emitting at longer wavelengths. In crude firefly extracts and some luciferin preparations, dehydroluciferin is usually present. It is a potent inhibitor of the light reaction. The chapter describes the method of collecting and drying fireflies in order to prepare an appropriate acetone powder, the preparations and properties of a crude and partially purified luciferase and finally the preparation of crystalline firefly luciferase.

Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase

The gene for firefly luciferase (luc) can be used as a generalized genetic probe. A method that aids in the analysis of shuttle vectors containing luc by allowing verification in Escherichia coli of a functional coding sequence is presented here. Colonies containing a functional form of luc are detected on film after luciferin is added to initiate the luminescent reaction. Two conditions, lowering the pH of the environment and maintaining aerobic conditions, were found to greatly improve the sensitivity of the assay. This technique may be useful in the development of genetic constructs that alter the natural coding sequence of luc, such as in gene fusions.

Factors affecting the kinetics of light emission from crude and purified firefly luciferase

Abstract Crude and purified firefly luciferase have been used to assay ATP from 0.2 pmol to 2 μmol. Over this range of ATP concentrations, there is a large change in the kinetics of light emission. At the lowest concentrations of ATP, light emission rises to a maximum and remains constant for a minute or longer. As the concentration of ATP is increased, the peak light intensity increases and the decay rate of light increases significantly. This is true for both the crude as well as the purified enzyme. High concentration of sodium arsenate as well as other salts inhibit the peak light emission and prevent the decay in light intensity which is due to product inhibition. It is possible to obtain almost any type of kinetics by manipulating the experimental conditions.

The production of oxyluciferin during the firefly luciferase light reaction

Abstract The luciferase-product complex (E · P) was isolated from the reaction mixture after light emission had occurred. The spectral properties of the product in the E · P complex are similar to those of oxyluciferin, with a broad absorption at 385 nm. The enzyme from the complex regains full activity upon the addition of substrates. The product is not covalently bound to the enzyme and readily dissociates in the presence of 6 m urea. The isolated E · P complex was found to have 1 mol of oxyluciferin per 100,000 daltons of luciferase. No AMP could be detected in the E·P complex unless inorganic pyrophosphatase was present during the reaction. In that case 1 mol of AMP per 100,000 daltons was found. Stopped flow studies showed that an increase in 385 nm absorption occurred concomitant with light emission. Measurement of the initial rate of product formation and the rate of photon emission showed they were identical, suggesting that oxyluciferin is indeed the light-emitting product. In the initial burst of the reaction two oxyluciferin moles per 100,000 daltons of luciferase are formed. A plot of the log of the initial rate of product formation was biphasic, indicating that the first mole of product is formed at a faster rate than the second. These results are consistent with previous experiments. However, they do not resolve the question of the molecular weight of the catalytically active species.

Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns.

Poly (A)+ RNA was isolated from the lanterns of adult fireflies, Photinus pyralis. The Poly (A)+ RNA was translated in a cell-free translation mixture from rabbit reticulocytes and the synthesis of enzymatically active firefly luciferase was demonstrated. The translation products were immunoprecipitated with anti-luciferase and then subjected to SDS gel electrophoresis. It was shown that a newly synthesized polypeptide exhibited an identical electrophoretic mobility as the purified enzyme.

Two kinetically distinguishable ATP sites in firefly luciferase

Results are presented which indicate that firefly luciferase has two catalytically active sites. One site, Km of 1.1 X 10(-4) M ATP, is responsible for the initial flash and is apparently product inhibited for further light production. The second site, Km of 2 X 10(-5) M ATP, catalyzes the continuous low production of light. ATP or AMP is a potent inhibitor of the initial flash when LH2-AMP is used to initiate the light reaction but appears to have no affect on the second site low level light emission. Both sites must be occupied by ATP for the formation of one L-AMP. Thus, ATP appears to function both as a catalytically active substrate and a regulator for light emission.

Immobilization of firefly luciferase on glass rods: properties of the immobilized enzyme.

Firefly luciferase has been covalently linked with glutaraldehyde to alkylamine glass beads which had been cemented to glass rods. The immobilized enzyme has a lower pH optima than the soluble enzyme and emits light with a major peak at 615 nm, while the soluble enzyme emits light with a peak at 562 nm. The immobilized enzyme is stable and can be used for multiple assays. The peak light intensity is linear with respect to ATP concentration in the range of 1 × 10−5 to 1 × 10−8m. The luciferase rods have been used in a coupled assay to measure the rate of ATP production catalyzed by creatine phosphokinase. This immobilized luciferase should be very useful for assaying low levels of ATP in any type of sample.

The creatine kinase curve area and peak creatine kinase after acute myocardial infarction: Usefulness and limitations

We determined creatine kinase (CK) curve areas in 112 patients with acute myocardial infarction. Two-hour sampling was performed for the first 24 hours or until peak CK was reached, and a gamma density function was used to calculate curve areas from all available samples. Attempts to predict CK curve area by means of the portion of the curve prior to peak CK proved to be inaccurate; not until values 2 hours or more beyond peak CK were utilized did predicted and actual CK areas agree well. A good correlation (r = 0.93) was found between CK area and peak CK. To establish an approach for detecting peak CK in the clinical setting, a range of sampling intervals (4 to 24 hours) was assessed; 4- and 6-hour sampling intervals for 48 hours produced maximum CK values at or above 85% of true peak CK in 90% and 89% of patients, respectively, and average maximum CK at both sampling intervals exceeded 94% of that obtained with 2-hour samplings. We conclude that this simplified approach can provide a basis for estimating infarct severity in the individual patient.

Developmental changes in creatine phosphokinase isoenzymes in neonatal mouse hearts

Abstract The distribution of creatine phosphokinase isoenzymes differs in extracts of newborn and adult mouse hearts. Electrophoresis on acetate strips reveals the presence of BB, MB, and MM isoenzymes in the 2 day old neonate heart, with relative activities of 4%, 24% and 72% respectively. Beginning at 6 days of age, a fourth isoenzyme, shown to be associated with mitochondria, is seen moving toward the cathode. With age the distribution changes, with BB disappearing by 18 days. By 25 days the relative proportions of MB, MM and mitochondrial CPK have reached 5%, 86% and 9%, respectively, similar to the levels seen in the adult. The late appearance of the mitochondrial isoenzyme may reflect a difference in the requirement of the developing and adult heart for ATP and phosphocreatine.

Creatine kinase isozymes in human tumors.

Abstract The creatine kinase content and isozyme distribtution in 33 human tumors carried in athymic mice has been determined. All of the tumors except two melanomas contained significant amounts of the BB isozyme, the mammalian fetal type enzyme, but very little of the MM isozyme. Many of the tumors of diverse origin contained a significant amount of the mitochondrial isozyme. Some of the serum from mice carrying these tumors showed elevated levels of either BB or mitochondrial creatine kinase, however, this does not seem to be specifically associated with any single type of tumor.