Detection of ATP and NADH A Bioluminescent Experience Ted C. Sellg: Kathryn Ann Drozda, a n d Jeffrey A. Evans2 University of Southern Mississippi, Hattiesburg, MS 39406 Everyone who has seen the flash of a firefly has observed t h e phenomenon of bioluminescence. Biological light production in various animals. olants. and hacteria has onlv recently heen understood in ~ o l e c u l &terms. Light produ&ion is dependent upon ATP in firefly tails (1)and reduced coenzymes such as NADH or FMNHz in some bacteria (2).These fmdigs f o m the basis for using bioluminescence as a sensitive analytical tool for detecting extremely small quantities of ATP (3. and NADH down to 10-l5and 10-l4moles.. resoectivelv . 4). Bioluminescent methods have wide-ranging utilit; for analysis; they have been used in detection of bacterial contamination, in determination of cell viability, in developing sensitive immunochemical assays, and in research areas where sensitive enzyme assays are required (3,5). Described below is a bioluminescent assay for ATP and NADH that meets the needs of an undergraduate biochemistry laboratory course. This experiment provides students with experience in hoth bioluminescence and analytical biochemistry yet requires limited instrumentation, preparation, and expense. T h e experiment as described can be run in one 3-hr time period and has potential for being used in conjunction with the measurement of metabolic levels of nucleotides and coenzymes. Theory Bioluminescence uses energy gained from enzymatic reactions to excite electrons from low energy levels into excited levels. It is thoueht that the excited state isachieved throueh the breakdown of an a-peroxylactone or peroxyflavin intermediate ifi.7). When these excited electrons return to their ground st& they emit light. By measuring the light given off, and bv kuowine the chemistw of the reactions involved. we can determine theamount of limiting suhstrate present. For the assavs we describe. either A'1'1' or NADH will he the limiting sul;strate. The enzvmatic reaction for the ATP assav is a two-steo process as shown below (8)
+
luciferin ATP
+
l"~ifrra%S
+
(1)
+ COz + AMP + light
(2)
lueiferyl-AMP PPi
l~~iflucifsrssalucifsrssalucifsrssa
luciferyl-AMP Oz +oxyluciferin Fireflv tail extract orovides hoth the luciferase and the luciferin (generic names for the enzymatic and nonenzymatic comoonents. resoectivelv). Addition of ATP to the extract leads to ligllt o k p u t proportional to the ATP concentration. T h e NADH assay is a coupled reaction (9). T h e lightevolving reaction is
0
FMNH,
+ O* + R-C-H II
luriferaue ----c
0
FMN 918
+ R-C-OHII
Journal of Chemical Education
+ light + H20
This reaction can he used to determine FMNHz levels, but to measure NADH we couple the above reaction t o NADH as follows NADH + H+FMN
PMN d u c t a e e
NAD+ + FMNH?
A partially purified bacterial luciferase contains sufficient FMN reductase to couole NADH to lieht oroduction. Addition of NADH to the hiiferase in the p k e n c e of excess FMN and decvlaldehvde (or similar lonechain aliphatic aldehyde) results in lighGrodudion propokonal t o i h e NADH concentration (10). T h e amount of light given off in each system can he measured by instruments, such as luminometers, scintillation counters, fluorometers, or UV-visible spectrophotometers, that contain sensitive photodiodes or photomultipliers. We chose t o work with aninexpensive Turner UV-visible spectrophotometer because of the likely availability of this instrument to most biochemical laboratories. With the lamp out and the analog output connected t o a recorder, one can measure the intensity of light emission with respect t~ time. The area under the light versus time curve is directly proportional t o both light output and concentration of limiting suhstrate (ATP or NADH). Experimental The solutions for the ATP assay were prepared in the following manner. Firefly lantern extract, Sigma FLE-50, was dissolved in 5 ml H20. This solution can be stored for up to 2 da refrigeratedor longer if frozen. Sigma FF-ATP, (1mg ATP, 40 mg MgS03 was dissolved in 100 ml HzO, producing approximately a 20 pM solution. This solution is stable when stored frozen. Actual concentratwns were determined rpettn~phoromerrically( A m 1 1 cm'\' = 15.1 X 10% I.ieht emission was initiated bv iniectine -aliauots . of [he ATP soluti& into 1.25 ml of lantern extrict,using a 50.~1syringe. The same lantern extract solution was used for each series of measurements. The light-emittingsolution for NADH was prepared by mixing 2.6 ml sodium phosphate buffer (0.1M, pH 6.8),0.2 ml mercaptoethanol (0.1% HzO), 0.4 ml Sigma 5 p e V bacterial luciferase (10 mglml buffer),0.4 ml FMN (0.2 mglml H20),and 0.1 ml decylaldehyde (1 mglml methanol). Care should he taken when working with mercaptoethanol and decylaldehyde, which is susceptible to oxidation. Mereaptoethanol is a toxic oompound;solutions should be prepared in a hood and kept covered. A 200 pM NADH solution was prepared by dissolving 1.8 mg NADH in 10 ml Hz0. Actual concentrations were determined spectrophotometrically (Am 61 ,IM = 18 X 109. AU solutions can be prepared in advance, with the exception of luciferase and FMN, which must be prepared fresh and protected from light. A 100-p1syringe was used to inject aliquote of the NADH solution into the light-emittingsolution. This same solution was used for each series of measurements. Light emission was ohsenred using a Turner 350 spectrophotometer with the lamp removed and the slit opening covered. A Varian G-15-1 strip-chart recorder was connected to the analog output of the spec-
' Present Address: University of Nebraska. Linwln. NE 68501. Author to whom inquiries should be sent.
trophatometer. With the sensitivity of the strip chart recorder set to 10 mV, a 1%transmittance reading on the spectrophotometer produced full-scale pen deflection. An injection port was improvised hv taping conductive black plastic over the sample compartment. ATP and NADH samnles were vieorouslv. iniected . throueh the ~lasticinto their respective liplht.emittinp,solutions. Immediately after injection the needle hole was coverd to reduce scattered light. Light output was rnonitorpd on the recorder until the signal decayed (ahout 4 5 rnin),and the area under the intensityversus time curve was measured by cutting and weighing the chart paper responses.
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Results and Discussion Iniection of the substrate throueh the lieht-tieht coverine into 'the luciferase-luciferin soluti& allowed oLervation 2 the entire light emission curve. The substrates ATP and NADH exhibited different emission versus time curves (Fig. 1). When ATP was injected into the solution there was a rapid increase in emission, reaching a peak in only 2 sec, followed by a decay. Injection of greater volumes of ATP resulted in higher emission peaks and slower decays as shown in Figure 1A. In contrast, NADH yielded a more gradual rise in intensity, requiring 6 see to reach a peak after injection, followed by a slower decay (Fig. 1B). As was found with ATP, addition of increased amounts of NADH resulted in increased peak heights and longer decay times. Total light output for each volume of ATP or NADH injected was calculated from the emission curves (Figure 1and additional substrate iniections). A ~ I ooft lieht outout versus ~ H rebealed a iinear Glationsh$ amount of ATP or N A ~ added hetween substrate concentration and light emission (Fig. 2). These linear standard curves allow quantitative determinations of unknown ATP or NADH concentrations. This experiment can be enhanced by giving students ATP and NADH unknowns. We have been able to use the ATP assay t o determine the ATP concentrations of red blood cells. A simple nucleotide extraction of packed red blood cells (11) and dilution of 1:1000 gave ATP concentrations within our range of measurement. Injection of several samples into the same luciferase-luciferin solution (Fie. 2) caused a net volume increase of onlv 7%. The concent~atfonchangeassociated with this volume increase would be sir?lif~cantin ~reciseanalvtical work: however, in a laboratory exercise it c& be neglected. The b"ild-up of products was not a problem over the course of several series &injections. ~njectionof 50 p1 of 3 mM AMP quenched the ATP reaction: therefore, an extreme excess of products would cause decreased emission. Optimal bioluminescent measurements were found to be dependent upon a number of factors. Nonconductive black plastic created a noise problem, possibly due to static electricity. The use of conductive plastic eliminated this problem. Another key to good results was vigorous injections of substrate into the light-emitting solutions in order to provide adequate mixing. Injections of very small volumes (5-10 p1) sometimes resulted in poor mixing and subsequently delayed light output making i t difficult to measure. Reduced lighting in the lab lowered the level of backeround noise durine injections. Luminescence is a sensitive function of temperature 14). For accurate results the lieht-emittine solution should he held a t a constant temperatuie. Other instruments were used t o Drovide a sensitivitv comparison. A Hitachi Model 100-60 ~pktrophotometereqkpped with an integrating sphere gave linear results a t 1000-fold lower concentrations of ATP and NADH than needed for the Turner. A Packard Tri-carb Scintillation counter showed sensitivity similar to the Hitachi. An American Scientific SPF 125 fluorometer also gave satisfactory results. The analog output of a Bausch and Lomb Spectronic 20 was found to be 10-100 times less sensitive than the Turner. The assays for ATP and NADH can be used together t o make a complete laboratory experiment or used independently to measure metabolic levels in a metabolism experi-
TIME (minutes)
Figure 1. Relative intensity versus tlme recordings lor inledions of (A) a 16 pM ATP soiutim: a. 5 pl; b. 20 pl; c. 30 pl; d. 50 pl; and (B) a 220 pMNADH solution: a. 10pl;b. 30 pl; c. 50 pl: d. 100 pI.
Figure 2. Plot of IigM aulput versur microliters of samplt, added fa (A) 16 pM ATP. and (B) 220 pMNADH. ment. The ATP assay alone is a simple, yet sensitive experiment requiring limited preparation and expense. These assays are simple to perform, easy to set up, and provide students with valuahle experience in a number of techniques. The modification of a spectrophotometer to read emission, the hook-up of the spectrophotometer to a stripchart recorder, and the use of cutting and weighing integration are analytical techniques not often encountered by undergraduates. The striking sensitivity of bioluminescence enables the student to see the potential of analytical biochemistry. We are pleased with this experiment and plan to make it a part of our undergraduate biochemistry laboratory curriculum. Volume 61 Number 10 October 1984
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Acknowledgment
We wish to thank Howard P. Williams for his helpful comments in preparation of this manuscript. Literature Cited (1) McElmy, W.D..Pm. Natl. Acad Sci. USA.Sl,WZ (1947). (2) Stan1ey.P. E.. Methad8 Enrymol., 57.215 (1978).
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(3) Stanley, P. E., "Biolumiosaeena and Chcmiluminawenee" (Editors: DcLuca, M. A,, and McElmy, M. D.) New York, 1981, p. 275. (4) Stanley. P. E..AWJ. Bimham., 39, MI (1971).
(5) Frieke,H., strasbugc., C. L a n d w m d , W. G.,J. CKW.them. c ~ i B n~~~~.~..ZO,SI 119R21. (6) Adam.W , J. CHUM.EDUC., 62,136 (1Yl5). (7) Hsatings, J. W. and Ne8hon.K. H.,Ann Re". Micmbiol..3I,M9 (1971). (8) hhningsr, A. L.."Princi~les of Biochcmistr,((. Worth. Nsn York, 198z.p. 388. (9) Hastings, W. J. and Tu,S.C.,Ann. N Y. Aeod. Sci., 366,316 (1981). (10) Wampler, J. E.,"Biolumingscenain Action (Editor: Hening.P. S.)AudemicPm, New York. 1978.n.11.
