A sensitive assay for flavin mononucleotide using the bacterial bioluminescence reaction

BIOCHEMICAL

MEDICINE

A Sensitive

1, 252-260

Assay

for

Bacterial EMMETT Goddard

(1967)

Flavin

Mononucleotide

Bioluminescence

W. CHAPPELLE, Bn'D ROBERT Space

Flight

Greenbelt,

December

the

Reaction

GRACE LEE H. ALTLAND’

Center,

Received

Using

PICCIOLO,

Ma.ryland

,“0771

19, 1967

We have been concerned for some time with the development of methods for the detection of extraterrestrial life suitable for use in unmanned space vehicles. In view of the ubiquitous occurrence of flavin compounds in terrestrial organisms, a system which appears promising is the bioluminescent reaction occurring in photobacteria, which specifically requires flavin mononucleotide (FMN) for light emission (1). The rationale and feasibility of this technique as a life detection system are presented elsewhere (2). This paper discusses the assay method with emphasis on medical and laboratory applications. We are able to define, using the bacterial bioluminescence reaction, the experimental conditions for a rapid and simple photometric measurement of reduced FMN (FMNH,) . Present techniques for the quantitative assay of this compound are relatively insensitive and slow. Fluorometry, which is perhaps the most sensitive, suffers from a lack of specificity. The interference of other compounds, such as chlorophyll (3), pterines (4), alloxan (5)) beneo[a]pyrene (6), etc., which have similar excitation and fluorescence maxima, would require that the FMN be isolated in a pure state. Table 1 presents TABLE SENSITIVITY

OF METHODS

1 FOR

FMN

ASSAY

Lower limit of detection (pg)

Method Paper chromatography Cytochrome c reductase Lactic oxidase (8) Fluorometry (9) Bacterial bioluminescence ’ Present

address

: National

(7) (7)

Institutes

0.01 0.01 1 0.0001

0.00001 of Health, 252

Bet,hesda,

Maryland.

BIOLUMINESCENT

ASSAY

FOR

FM??

253

a comparison of the presently available techniques and their respective sensitivities for FMN assay (7-9). The reactions involved in bacterial luminescence have been the subject of intensive study by a number of investigators. The results of these studies are described in the most recent review by Hast,ings (10). A simplified overall reaction sequence for the in z~itro reaction is: luciferase

FMNHz

+ RCHO

2 + 02 -

light

+ products

The reaction intermediates and products are not yet definitely established although such studies are underway in this laboratory as well as elsewhere. In this reaction, the light being emitted is a measure of the reaction rate. It therefore follows that the greatest light intensity will occur at t,he time of the greatest number of simultaneous collisions between the neceesary components of the reaction. One can assume that in a multicomponent. reaction, with all components except one in large excess, the reaction rate (light intensity) should be directly proportional to the concentration of the limit,ing component. MATERIALS STOCK

AND

METHODS

S0LUTI0Ns

Luciferase. A lyophilized crude extract of Achromobacter fischeri (Sigma Chemical Co., St. Louis, Missouri) was dissolved in 0.05 M Trip buffer, pH 7.4, to a concentration of 1 mg/ml.3 The solution was stored in an ice bath until shortly before use, at which time it was allowed to reach the ambient temperature. The optimal reaction temperature is approximately 25” ; the enzyme is stable at, room temperature for approximately 1 hour. Dodecybaldehyde. The bisulfite addition complex of the nldehyde was prepared by adding 2 ml of dodecylaldehyde (K and K Laboratories, Inc., Plainview, New York) to 190 ml of saturated sodium bisulfite. After precipitation with 5 ml of methanol, the addition complex was washed with three 50-ml volumes of ether and air-dried. A saturated solution of the aldehyde addition complex was prepared by adding 100 mg of the addition complex to 100 ml of 0.05 M Tris buffer, pH 7.4, and shaking for 30 minutes. The undissolved material was removed by centrifugation. This solution is stable for about 8 hours. ’ Saturated long-chain aldehyde. 3To be described in a subsequent scale production of luciferase from

paper; we have developed a technique photobacteria grown on apar surfaces.

for large

254

CHAPPELLE,

PICCIOLO, AND ALTLAND

Sodium Borohgdride (NaBH,).

This compound, used in the dry state: and must be stored absolutely dry until weighed out. Palladium ChZoride (PdCl,).4 A stock solution containing 0.01 mg/ml in 0.05 M Tris buffer, pH 7.4, is stable indefinitely when stored in the cold. Flavin Mononucleotide. A stock solution of FMN (Sigma Chemical Co., St. Louis, Missouri) at a concentration of 1 mg/ml in 0.05 M Tris buffer, pH 7.4, is stable up to 1 month if kept in the dark and cold. is hydrophilic

INSTRUMENTATION

AND OPERATIONAL

PROCEDURE

Figure 1 is a schematic of the system used to measure light emission. The reaction chamber, attached to the photomultiplier tube housing, consists of a rotary cylinder mounted in an aluminum block. A section of t.he cylinder is cut out to accommodate a 6 X 50-mm glass cuvette. Imr-

HANDLE SYRINGE

DARK CURRENT 8ALANCE

INJECTION

PORT

PROPORTIONAL CUVETTE

T O PHOTOMULTIPLIER

FIG. 1. A schematic description

of the instrumentation

used for light measmxments.

mediately above the cuvette holder is a small injection port through which FMN is injected by needle and syringe into the enzyme solution. The signal from the photomultiplier tube (RCA lP21) is amplified, and the d-c signal from the amplifier (Photovolt 720SP) is read out on a chart recorder. Figure 2 shows a typical response curve. The response’ is not immediate and implies a rate-limiting ,step in the reaction. Data are presented in terms of peak height (maximum light intensity). Prior to use, two parts of the luciferase solution are mixed with one part of the aldehyde solution. Flavin mononucleotide is made up to the desired concentration in the PdCl, solution. The mixture of the luciferase solution and the aldehyde solution may be lyophilized and stored in the cold (-20”) in a desiccator where it remains stable indefinitely. After *Prtlladium

chloride

catalyzes the reduction

of FMN

by NaBHI.

BIOLUMINESCENT

ASSAY

TIME

FIG. 2. A typical containing

aldehyde

FOR

255

FMN

iSECl

light emission curve when and bacterial luciferase.

FMNHS

is injected

into

a solution

the addition of H,O to return it to its original concentration, reduction of the FMN is carried out by the addition of 10 mg of NaBH, to 10 ml of the FMN solution. The solution is allowed to stand between 10 and 15 minutes, after which a O.l-ml aliquot is injected into the reaction cuvette which contains 0.3 ml of the luciferase-aldehyde solution. RESULTS AND DISCUSSIOS Luciferme Concentration. Since we have assumed that the maximum light emission occurs when the enzyme is in excess, one would expect an increase in initial light intensity with increases in the luciferase concentration. This is demonstrated in the experiment described in Fig. 3. The

FIG. 3. The effect response luciferase

of the concentration of bacterial luciferase on the peak height in to the injection of 1 x lo-’ PR of FMNH,. The buffer solution in which the was dissolved contained 1 mg of bovine serum albumin/ml.

256

CHAPPELLE,

PICCIOLO,

AND

ALTLAND

use of 1 mg/ml of enzyme should ensure an excess of enzyme with respect to the FMN concentration that one expects to encounter in most biological materials. Dodecybaldehyde. The hisulfite addition complex, a solid which can be made and stored indefinitely under proper conditions of desiccation, is not, as susceptible to oxidation as the free aldehyde. -41~0, more accurate quantitation is possible. There is very little difference in its solubility compared with the free aldehyde. In fact, before it becomes active in the system, the free aldehyde must be split off. Studies by Hastings (11) have shown that, the light emission during the bioluminescent reaction is increased with increase in the aldehyde chain length up to a maximum of 14 carbons, although the studies reported here hare been done with the 12-carbon aldehyde. So&l?)& Borohydride. The NaBH, concentration is critical (Fig. 4). We do not understand the reason for this at the present time, but enzyme inhibition is suspected.

Na@ti,

FIG.

4. The

concentrations

peak height of NaBHa.

observed

when

(mg/mii

1 pg of FMN

was

reduced

with

varying

Palladium Chloride. Light emission as the result of changes in the PdCl, concentration is essentially the same over a range extending from 0.1 to 0.0001% in the final reducing mixture, which eliminates its concentration as a critical factor. Light Emission. as Function of FAIN Concentration. The relationship between the FMN concentration and the initial light intensity was analyzed statistically by polynomial regression. The best fit curve (Fig. 5) is the linesr regression (light units = 1.053 FMN + c) with first-order regression significant at the 0.1% level. Therefore, a st,atietically valid

BIOLUMINESCENT

gG-5

ASSAY

1 -4

FOR

I -2

/ -3

257

FMN

.-A -1

LOG IpeFMNH2)

FIG. 5. Statistical regression line illustrating as a function of the FMN concentration.

the linearity

of the initial

peak

height

linearity exists between the initial light intensity and FMN from 10-l to 10m5 pg/O.l ml. Preparation of an FMN concentration curve for each experiment should be routine due to variation in enzyme activity from batch to batch. It is evident from the concentration curve (Fig. 6) that solutions containing FMN at a concentration above 0.1 pg/O.l ml must. be diluted to a concentration which falls on the linear portion of the curve. Endogenous Light. In carrying out this assay with unpurified enzyme, there is light emission of extremely low intensity in the absence of a reducing agent. This is attributed to the presence of DPNH dehydrogen-

51

-6

I

’

-5

I

I

-4

-3

,

-2

I

-1

I

I

0 069'

LOG h8FMNH2)

FIG.

6. Peak

height

rto

as a function

of the

concentration

of FMN&.

258

CHAPPELLE,

PICCIOLO,

AND

ALTLAND

ase, as suggested by Kuwabara et al. (12). Further, upon the injection of NaBH, in the presence of PdCI,, a fairly low-level light emission occurs. We currently interpret both of these phenomena as resulting from the presence of endogenous FMN which is subsequently reduced by the NaBH, or the DPNH dehydrogenase. The intensity of the endogenous light is variable and is a function of the history of the enzyme being used. Flavin mononucleotide measurements are corrected for these endogenous light emissions. Table 2 describes representative assays of certain biological materials. The FMN was extracted by incubation of the organisms for 2 minutes with 6% butanol in 0.01 M Tris buffer containing 1 X 10V3M EDTA. This has been found thus far to be the most effective agent for extraction. TABLE FMN

CONCENTRATION

2

IN BIOLOGICAL

Material Escherichia coli Serratia murcescens Bacillus globigii Bacillus globigii spores

Urine Whole blood (5.5 X lo6 rbc/mm3) Serum

MATERIALS

fig/cell” or ml 7 x

lo-”

6 X .lO-‘2 3 x 10-n 3 x lo-” 1 x

lo-”

5.0 1.1

(1Organisms were assayed during their stationary phase of growth, and cell number was determined by a plate count on tryptic soy agar.

It is of great interest in the case of Bacillus globigii spores that the FMN concentration is the same as that found in vegetative cells. This differs from the observation which we made previously with regard to ATP determination in spores versus vegetative cells (13). The presence of two orders of magnitude more ATP in vegetative B. globigii cells than in B. globigii spore cells could be interpreted as a decrease in the requirements for energy necessary for the low metabolic activity occurring m spores, with the total FMN concentration remaining constant. Since most of the riboflavin accumulated by mammalian organisms is excreted via urine, a measurement of FMN of urine should be of interest. A urine specimen of a normal male in good health contained 1 X 10-l pg/ml. This is consistent with the results published by Morrell and Slater (14)) although these investigators were determining the total riboflavin content of urine. It is therefore thought that this rapid, simple method will be of great advantage in the evaluation of metabolic disorders involving flavin metabolism.

BIOLUMINESCENT

ASSAY

FOR

FMN

259

There are additional implications in the data presented here, which are not related to the use of the bacterial luminescence system as an assay procedure. The linearity of light emission in response to the FMN concentration may prove valuable in resolving the question of quantity and physical state of the reduced FMN participating in the light-emitting reaction. Our use of the reducing agent NaBH4 may result in t,he production of a different form of reduced FMN, i.e., FMNH,, FMNH,, or FMNH,, than the DPNH dehydrogenase reaction. Investigations along these lines are in progress. Preliminary measurements of the quantum yield gave a value of lo-“lo4 for FMN. The quantum yield measurement was done by integration of the light resulting from a known quantity of FMN based on a calibrated light standard. This value differs considerably from that reported by Lee and Seliger (15). Possible reasons for this discrepancy are the difference in reductant used and a possible deterioration in the light standard (16). The linearity of response observed in the experiments reported here, and a titration of the degree of reduction of FMN by NaBH,, may resolve pertinent questions regarding the phyeicochemical mechanism of bacterial bioluminescence. In conclusion, we would like to stress that in the characterization of many enzyme systems, a knowledge of the FMN concentration as well as that of FAD is of great importance. As has been shown by other investigators (17)) it is not a difficult procedure to convert FAD to FMN. Of equal and possibly greater significance from a clinical standpoint is that this is a technique that could be used in the evaluation of riboflavin deficiencies in certain metabolic disorders. SUMMARY

Investigations of the feasibility of the bacterial bioluminescence reaction as the basis of an extraterrestrial life detection system have resulted in the development of a photometric technique for the quantitative detection of 10-l’ gm of flavin mononucleotide (FMN). The FMN, reduced with sodium borohydride and palladium chloride in solution, is a required component. of the in vitro bacterial bioluminescent reaction. Under the conditions of the procedure, we have found a linear relationship between light intensity and concent8ration of reduced FMN. The concentrations of FMN in bacteria, urine, and blood have been determined. ACKNOWLEDGMENTS We thank Drs. Norman H. MacLeod and Howard Seliger t,ions during the course of these investigations, and Dr. consultation in statistical design and analysis. The technical Yates is gratefully acknowledged. Dr. J. W. Hastings kindly scintillator for standardization measurements.

for their helpful suggesEdward Batschelet for assistance of Mrs. Janice provided the radioactive

260

CHAPPELLE,

PICCIOLO,

AND

ALTLAND

REFEREXCES 1. MCELROY, W. D., HASTINGS, 118, 385 (1953). 2. PICCIOLO, G. L., CHAI’PELLE, 3. FRENCH, C. S., AND YOUNG, 4. SIMPSON, D. M., Analyst 72, 5. ARCHIBALD, R. M. J., J. Btil. 6. SAWAKI, E., HAUSER, T. R., (1960). 7. HUENNEKENS, F. M., AND

8.

9. 10. 11. 12. 13. 14. 15. 16.

17.

J. W.,

SONNENPELD,

V., ;ISD COULOMBI~E,

L.; Sciel/cc

E. W., AND RICH, E., BioScience, in prces V. K., J. Gen. Physiol. 35, 873 (1952). 382 (1947). Chem. 157, 507 (1945). AND STANLEY, T. W., Intern.

J.

iiir Pollution

(1968).

2, 253

FELTON, S. P., i)z “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 950, p. 957. Academic Press, New York, 1957. BERGSIEYER, H. (Ed.), in “Methods of Enzymatic Analysis,” p. 599. Academic Press, New York, 1965. BURCH, H. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 960. Academic Press, New York, 1957. HASTIX~S. J. W., in “Current Topics in Bioenergctics” (D. R. Sanndi, ed.), Vol. 1, p. 113. Academic Press, New York, 1966. HASTINGS, J. W., GIBSON, Q. H., FRIEDLAND, J., AND SPUDICH, J., in “Bioluminescence in Progress” (F. H. Johnson and Y. Haneda, eds.), p. 151. Princeton Univ. Press, Princeton, N. J., 1966. KUWABARA, S., CORMIER, M. J., DURE, L. S., KREISS, P.. AND PFUDERER, P., Proc. Natl. Acad. Sci. U. S. 53,822 (1965). CHAPPELLE, E. W., AND LEVIN, G. V., Rio&em. Med., in press (1968). MORRELL, D. B., .~ND SLATER, E. C., Biochem. J. 40, 652 (19461. LEE, J., AND SELIGER, H. H., Photochem. Photobiol. 4, 1015 (1965). REYNOLDS, G. T., LIUZZI, A., HASTINGS, J. W., STRUNK, B. L., .~ND REYNOLDS, T., Biol. Bull. 133, 480 (1967). UDENFRIEND, S., ‘LFluorescence Assay in Biology and Medicine.” Academic Press,

New York, 1962.