Proc. Nati. Acad. Sci. USA Vol. 77, No. 1, pp. 249-252, January 1980 Biochemistry Physical interaction and activity coupling between two enzymes induced by immobilization of one (bacterial bioluminescence/luciferase/flavin reductase/flavoproteins/monooxygenase) SHIAO-CHUN Tu* AND J. WOODLAND HASTINGS The Biological Laboratories, 16 Divinity Avenue, Harvard University, Cambridge, Massachusetts 02138 Communicated by E. R. Blout, October 15, 1979 ABSTRACT Flavin reductase and bacterial luciferase are to provide a partially purified flavin reductase, which exhibited believed to be coupled in the in vivo light emitting reaction. In an activity of 0.21 unit/mg; 1 unit is defined as the oxidation extracts, however, they are both soluble enzymes that exhibit little or no association. Immobilized luciferase, covalently at- of 1 ,mol of NADH at 23°C in 1 ml of 0.05 M phosphate, pH tached to Sepharose, was found to bind the soluble reductase 7/50 jAM FMN/0.2 mM NADH. Cyanogen bromide and and to exhibit activity in the coupled reaction with an enhanced Sepharose 6B were products of Eastman and Pharmacia, re- efficiency of electron transfer. spectively. FMN and NADH were obtained from Sigma. Luciferase activity was determined in a standard assay Bacterial luciferase utilizes reduced flavin mononucleotide (nonturnover) as the initial maximum light intensity upon the (FMNH2) as its substrate, a compound that is highly autoxi- injection of 1 ml of 50 pM FMNH2 (catalytically reduced) into dizable (1, 2). In cell extracts there are enzymes (reductases) 1 ml of 0.05 M phosphate (pH 7) containing 0.2% bovine serum capable of producing FMNH2 from NAD(P)H In flavin reductase albumin, 0.001% decanal, and luciferase. this nonturnover NAD(P)H + H+ + FMN - NAD(P)+ + FMNH2 assay, all of the luciferase-flavin intermediate is formed within the first second, at which time the bioluminescence intensity FMNH2 + 02 + CH3(CH2)nCHO (light-emitting rate), in quanta sec-1, reaches a maximum (IO). luciferase Light intensity then decreases exponentially; the first-order rate -* light + FMN + H20 + CH3(CH2)nCOOH constant of this decay provides a measure of the catalytic rate. The reduced flavin produced in vitro by the reductase is free In the coupled assay, the bioluminescence is initiated by the and subject to autoxidation (3, 4). But it has always seemed to addition of flavin reductase to 1 ml of 0.05 M phosphate (pH us that in vivo there must be some complex between the re- 7) containing luciferase, 0.2% bovine serum albumin, 0.001% ductase and luciferase such that the cellular supply of reduced decanal, 50 ,uM FMN, and 0.25 mM NADH. In the coupled pyridine nucleotide would not be subject to uncontrolled dis- assay, both enzymes turn over. Luminescence was measured sipation via a shunt leading to freely autoxidizable flavin and at 23°C with a calibrated photomultiplier photometer (11). H202 production (5). One report suggested the existence of a Luciferase was immobilized on Sepharose 6B by using the luciferase-flavin reductase complex, but there is still little ev- method of cyanogen bromide activation (12). Sepharose 6B gel, idence for specific interaction between these two enzymes about 5 ml of bed volume, was washed by filtration with 200 (5-7). ml of water and suspended with constant stirring in an equal Although both luciferase and reductase in cell lysates are volume of water; 1.25 g of cyanogen bromide was added over soluble and not associated with a particulate fraction, there is a period of 20-30 min. During this step, the temperature was evidence suggesting that luciferase functions in vivo in associ- kept between 18 and 22°C by the addition of ice and the pH ation with membrane proteins (8). As a model for the mem- was kept at 10.5-11.5 by the addition of 2.5 M NaOH. The brane-bound enzyme system, we covalently attached luciferase activation of gel is about complete when no more base is needed to Sepharose 6B and examined the interaction of the immo- to maintain the desired pH. The suspension was immediately bilized luciferase with soluble flavin reductase. The results in- cooled to 4°C, filtered, washed with 200 ml of precooled 0.1 dicate that both binding and electron transfer between the two M phosphate (pH 7.5), and suspended in 5 ml of the same proteins are enhanced with the immobilized luciferase. precooled buffer containing 10 mg of luciferase. After gentle MATERIALS AND METHODS shaking at 4°C for about 18 hr, the suspension was washed by centrifugation at 0°C five times in 10 ml of 0.1 M phosphate, Luciferase was purified from Beneckea harveyi strain 392 (9) pH 7. After each centrifugation, the supernatant was collected by the method of Gunsalus-Miguel et al. (10) to a specific ac- for determination of luciferase activity, and protein content was tivity of 1.7 X 1014 quanta/sec per mg of protein determined measured by the method of Lowry et al. (13). This allowed us at 230C by the standard FMNH2-initiated assay (see below). to the amount of bound A NADH-dependent flavin reductase activity was resolved calculate by difference luciferase; this from luciferase activity at the stage of DEAE-Sephadex column value is used in estimating the specific activity of the immo- chromatography. The peak activity fractions were combined bilized luciferase. For experimental studies, the thoroughly washed gel was suspended in an equal volume of 0.1 M phos- The publication costs of this article were defrayed in part by page phate, pH 7/0.1 mM dithiothreitol and stored at 0°C. charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Department of Biophysical Sciences, University of this fact. Houston, Houston, TX 77004. 249 Downloaded by guest on September 26, 2021 250 Biochemistry: Tu and Hastings Proc. Natl. Acad. Sci. USA 77 (1980) Table 1. Properties of soluble and immobilized luciferases its catalytic rate with the 10-carbon aldehyde (decanal) was somewhat (20%) slower. Parameter* Soluble Immobilized Thus, the decreased activity cannot be attributed to de- Relative specific activity, % 100 4 creased catalytic efficiency. The lower activity might be due Catalytic ratet min- to quenching of excited states produced in the reaction. Alter- Octanal 2.4 2.3 natively, 95% of the attached molecules might be catalytically Decanal 17.8 14.1 inactive. Luciferase is a heterodimer designated af3 (15, 16); Dodecanal 2.4 2.2 a is involved in catalysis, whereas the specific function of 3 is Denaturation temperatures 0C not known (4). Because luciferase activity is much more sensi- 0.05 M phosphate 44 48 tive to modifications in a than to those in : (17-20), inactive 0.75 M phosphate 55 57 attached luciferase might be attributable to the site on the Energy of activation for thermal protein at which the covalent link is made. inactivation, kcal-mol'1 In addition to its decreased activity, the immobilized lucif- 0.05 M phosphate 64 28 erase is also different from the soluble enzyme with regard to 0.75 M phosphate 89 24 its thermal stability. Heat denaturation of immobilized lucif- * All activity measurements were made by using the FMNH2-initiated erase in phosphate buffer required a higher temperature and standard assay at pH 7 and 230C. t Luminescence decay rate at 230C in the nonturnover standard exhibited a much lower energy of activation (Table 1). As assay. shown, both of these parameters were sensitive to ionic strength Temperature at which half the activity is lost in 5 min. or phosphate concentration, but the immobilized luciferase was distinctly different from the soluble form in its stability prop- erties under all conditions examined. RESULTS AND DISCUSSION When partially purified flavin reductase (NaDodSO4 gel, The properties of immobilized luciferase are shown in Table see Fig. 1A) was mixed for 5 min at 00C in 0.05 M phosphate 1. Its apparent specific activity was lower than that of the sol- (pH 7) with the luciferase immobilized on Sepharose 6B, about uble enzyme by a factor of about 20, but its kinetic properties, 75% of the reductase activity was bound, as determined by the as judged by the decay rate in the nonturnover assay, were not reductase activity remaining in the five washes. Comparison greatly altered. Depending upon the chain length of the alde- of the patterns of NaDodSO4 gel electrophoresis of the material hyde used in the reaction, there are considerable differences solubilized from the Sepharose 6B-luciferase before (Fig. 1B) in turnover time with soluble luciferase (14, 15). Similar dif- and after (Fig. 1C) complexing with flavin reductase shows that ferences were exhibited by the immobilized luciferase, although relatively few of the proteins from the array of those present A .1 0.2 - "To 0.1 - 1 I ] 0.5 RF FIG. 1. NaDodSO4 gel electrophoresis ofthe partially purified soluble flavin reductase (A), proteins solubilized from Sepharose 6B-immobilized luciferase (B), and the immobilized luciferase complexed with flavin reductase (C). (A) Flavin reductase (50 ,g) was incubated in 0.1 ml of 0.01 M sodium phosphate, pH 7/1% NaDodSO4/1% 2-mercaptoethanol at 371C for 2 hr and then applied to a gel for electrophoresis. (B) Sepharose 6B-immobilized luciferase (0.4 ml gel volume) was similarly incubated in 0.4 ml ofthe buffer described above. (C) Immobilized luciferase complexed with flavin reductase and incubated as in B. After centrifugation, 0.1 ml of each supernatant was used for electrophoresis. Bands I, and 12 (B and C) were impurities in the luciferase sample used for the immobilization.