William D. McElroy

Spectral emission and quantum yield of firefly bioluminescence

1. 1. The emission spectrum of in vitro firefly bioluminescence has been found to be identical with the in vivo bioluminescence. 2. 2. One light quantum is emitted for every luciferin molecule oxidized, the yellowgreen emission band peaking at 562 mμ. 3. 3. A low quantum yield red emission appears in acid pH and at high phosphate buffer concentrations even at neutral pH. 4. 4. An energy stabilization role is postulated for the luciferase molecule in view of the high quantum yields measured.

[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.

Crystalline firefly luciferase.

Abstract 1. 1. The method for purification and crystallization of firefly luciferase is described. 2. 2. The crystalline enzyme is apparently homogenous as evidenced by ultracentrifugation a and electrophoretic determinations. The molecular weight is probably around 100,000 and the isoelectric point between pH 6.2 and 6.3. 3. 3. The effect of temperature, pH and the concentration of luciferin Mg ++ and ATP is described.

MECHANISM OF BIOLUMINESCENCE, CHEMI‐LUMINESCENCE AND ENZYME FUNCTION IN THE OXIDATION OF FIREFLY LUCIFERIN*,†

Abstract— The chemical steps and the products of the bioluminescent and chemiluminescent oxidations of firefly luciferin are elucidated. The colors of firefly bioluminescence can be explained in terms of different ionic excited states and spectral shifts due to changes in molecular environment. Firefly luciferase undergoes conformational changes during catalysis. There are two sites for light production per 100,000 mW. A regulatory mechanism involving dehydro‐luciferin is proposed for control of firefly flashing.

The function of coenzyme A in luminescence

Abstract Firefly luciferin (C13H12N2S2O3) reacts with ATP to form active luciferin (apparently adenyl-luciferin) and pyrophosphate. The oxidation of active luciferin leads to light emission and adenyl-oxyluciferin, the latter compound eventually decomposes into adenylic acid and oxyluciferin (C13H10N2S2O3). Oxyluciferin is a potent inhibitor of the light reaction and once it has reacted with ATP and luciferase, the latter is incapable of catalyzing the oxidation of luciferin. Coenzyme A stimulates light emission by removing oxyluciferin from the enzyme surface. The evidence indicates that oxyluciferyl-CoA is formed, which can react non-enzymically with cysteine, glutathione or hydroxylamine to form the corresponding oxyluciferyl derivatives. Chromatographic, isotopic and fluorometric data are presented to support the above conclusions. Oxyluciferyl-CoA in the presence of luciferase can be split by adenylic acid and when excess pyrophosphate is added ATP and free oxyluciferin are formed. The incorporation of 14C-adenylic acid into ATP depends upon the presence of CoA in the reaction mixture. The importance of these various reactions for light emission and electron transport is discussed.

Substrate-binding properties of firefly luciferase: I. Luciferin-binding site

Abstract Various techniques were used for characterization of the luciferin-binding site of the firefly luciferase. Both equilibrium-dialysis and fluorescence-titration techniques revealed the existence of two identical, noninteracting binding sites for dehydroluciferin, which is a potent competitive inhibitor. Kinetically obtained dissociation constants of various luciferin analogues and other competitive inhibitors revealed that most of the binding energies of these compounds reside in the backbone ring structure and that various substituents did not influence the binding to any significant extent. Exceptions to this are the methyl group at the 6-position of the benzothiazole ring and the carboxylic acid group. The binding energy of the luciferin backbone structure to the luciferin-binding site is 7.5 kcal/mole. Two parts of the luciferin molecule, the benzothiazole ring portion and the thiazoline ring portion, contribute 6.0 kcal/mole and 1.5 kcal/mole respectively to the binding energy. Comparison of the binding energies of several heterocyclic compounds structurally related to benzothiazole suggests that the nitrogen atom of the benzothiazole ring may be important in orienting the compound to a fixed position in the luciferinbinding site. The binding studies with N -ethylmaleimide-inactivated luciferase gave further evidence that the two sulfhydryl groups essential for the luciferase activity are located at the luciferin binding sites, most likely one at each binding site. Excitation spectra of luciferase-bound dehydroluciferin indicated that the phenolic group of dehydroluciferin is in unionized form on the enzyme even in a medium of high pH. This effect and a large enhancement of the blue fluorescence peak at 440 mμ suggest the environment of luciferin-binding site to be quite hydrophobic in agreement with earlier observations.

Substrate-binding properties of firefly luciferase. II. ATP-binding site.

Abstract Kinetically the MgATP complex was shown to be the substrate in the light reaction catalyzed by the firefly luciferase. Kinetic as well as inhibition studies showed that uncomplexed ATP is also bound to the luciferase, and is a competitive inhibitor with respect to MgATP. Similar studies for Mg 2+ indicated that it is not bound to the luciferase by itself. Equilibrium dialysis showed that 1 mole of MgATP and 2 moles of ATP are bound per 100,000 molecular weight of luciferase. Kinetic inhibition studies with ATP analogs showed that the 6-amino group of adenine is important for the binding of bases and nucleosides at the MgATP site. Energetically, adenine and negative charges on phosphate groups contribute 57% and 43%, respectively, toward binding (total binding energy = 4.8 kcal). dATP can also serve as a substrate in the formation of adenylate of both luciferin and dehydroluciferin. It is a poor substrate, having maximal velocity of light reaction only 5% that of ATP and equilibrium constant of dehydroluciferyladenylate formation five times smaller than that with ATP. Many divalent metal ions can replace Mg 2+ in the light reaction. Mn 2+ and Mg 2+ are the two most effective metal ions. Complexing of divalent metal ions larger than Mg 2+ with the adenine ring seems to interfere with the binding of such complexes to luciferase.

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.

Function of adenosine triphosphate in the activation of luciferin.

Abstract 1. 1. Using crystalline firefly luciferase and purified luciferin, it is possible to show that light production is associated with the utilization of both luciferin and ATP. The total light produced is directly proportional to the concentration of these two substrates. During luminescence there is a decrease of the 330 mμ absorption peak of luciferin. 2. 2. The reaction of ATP with luciferin leads to the formation of pyrophosphate and active luciferin (presumably adenylluciferin). The latter compound can either react with oxygen for light production or be hydrolyzed to luciferin and adenylic acid. The latter reaction occurs under anaerobic conditions. Adenylic acid has been identified as one of the products. 3. 3. The initial reaction of luciferin with ATP is reversible since the addition of PP32 to the reaction mixture leads to the formation of labeled ATP. Luciferin is required for this exchange. 4. 4. Oxidized luciferin as well as other derivatives of luciferin will replace luciferin as far as pyrophosphate liberation is concerned. In the presence of inorganic pyrophosphatase there is therefore a rapid and complete hydrolysis of ATP to adenylic acid and inorganic phosphate. The results indicate the labile nature of the adenyl compounds. 5. 5. Adenosine tetraphosphate, uridine triphosphate, cytidine triphosphate, guanosine triphosphate and inosine triphosphate are inactive for both light production and pyrophosphate liberation. 6. 6. The results are discussed in relation to the mechanism of light emission and the mode of action of pyrophosphate, pyrophosphatase, and coenzyme A on luminescence.

Anion inhibition of firefly luciferase.

Abstract The activity of firefly luciferase was shown to be very sensitive to the presence of salts in the assay mix. The general ionic strength effect was expressed by an increase in the Km for MgATP as the ionic strength was increased. When this effect was controlled by using concentrations of Mg2+ and ATP 10 times that normally saturating, a specific anion inhibition was observed. The order of effectiveness of inhibition by the anions, SCN− > I− ~ NO3− > Br− > Cl−, followed their position in the Hofmeister series. Only one anion was bound per active site and all the anions bind at this same site. Every reaction catalyzed by luciferase, even those proceeding by apparently different mechanisms, was affected in a similar manner by the anions. It was assumed that a small localized conformational change was occurring in the area of the active site upon the binding of the anions.