Separation of bacterial luciferase from oxidoreductases by affinity chromatography

ANALYTICAL

BIOCHEMISTRY

137-141 (1985)

151,

Separation

of Bacterial Luciferase from Oxidoreductases by Affinity Chromatography TENLIN S. TSAI

Packard

Instrument

Company,

United

Technologies,

Downers

Grove,

Illinois

60515

Received April 22, 1985 The NADH-dependent and NADPH-dependent oxidoreductase activities associated with bacterial luciferase in Vibrio (Beneckea) harveyi can simultaneously be removed from purified luciferase through a Blue Sepharose CL-6B column. The result achieved with this one-step affinity chromatography is similar to that obtained with two sequential “reverse-affinity” chromatographies. 0 1985 Academic

Press, Inc.

WORDS:enzyme purification; bacterial luciferase; oxidoreductase; bioluminescence; affinity chromatography. KEY

Bacterial luciferase (BL)’ catalyzes a bioluminescent reaction (reaction [b]) involving the oxidation of reduced flavin (FMNHJ and a long-chain alphatic aldehyde (RCHO) by molecular oxygen to yield the corresponding FMN, H20, acid (RCOOH), and light (l-3). The oxidation of NADH or NADPH by oxidoreductase produces FMNH2 (reaction [a]), which can in turn be used as the substrate for the luciferase reaction (4,5). The following reaction schemes were therefore established:

Since both NADH:FMN oxidoreductase (D’ase) and NADPH:FMN oxidoreductase (P’ase) are present in minute amounts in Vibrio (Beneckea) harveyi (6) and because of their

strong association with the BL, isolation of BL devoid of D’ase and P’ase is difficult, yet is essential in studying the induction of luciferase and reductases and their interaction in regulating bacterial bioluminescence. The NADHand NADPH-specific oxidoreductases have been purified with apparent homogeneity from bioluminescent bacteria with the use of affinity chromatography at the final isolation step (7,8). The properties and purification of bacterial luciferase have been described (9,lO) but the purified enzyme has not been evaluated for its contaminating D’ase and/or P’ase activity. Therefore the purpose of this study is to develop a simple and effective procedure to purify BL to contain minimal oxidoreductase activities. Two sequential “reverse-affinity”* chromatographic procedures were used at the final purification steps to bind D’ase and P’ase effectively and to result in a BL preparation containing less than 0.0 1% oxidoreductase activity. Similar results can be achieved with the simple one-step Blue Sepharose CG6B affinity chromatography.

’ Abbreviations used: BL, bacterial luciferase; alkanal, reduced-FMN:oxygen oxidoreductase ( 1-hydroxylating, luminescing), EC 1.14.14.3; D(P)‘ase, NAD(P)H:FMN oxidoreductase, EC 1.6.8.1; DTT, DLdithiothreitol, Cleland’s reagent.

2 Reverse-affinity chromatography: the contaminating enzymes, rather the enzyme of interest, have strong affinity for the column gel. The usage of the term “reverse” is further rationalized in the third paragraph of Results and Discussion section.

NAD(P)H

+ H+ + FMN Nz NAD(P)

FMNH:!

+ FMNH2

[a]

B.WtUial

+ O2 + RCHOluciferase FMN + RCOOH

+ H20 + light

[b]

137

0003-2697/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reaxd.

138

TENLIN

MATERIALS

AND

METHODS

S. TSAI

substrate) activities in BL were assayed by the coupled bioluminescent reaction ([a] and [b]). Chemicals and reagents. Most of the chro- The reaction was initiated by introducing 20 matographic gels such as DEAE-Sephadex A- ~1 of 100 PM NADH or NADPH to 20 ~1 of 50, Sephadex G-100, 5’-AMP Sepharose 4B, enzyme sample and an assay mixture conand Blue Sepharose CL-6B were obtained taining 0.1 ~1 of Decanal reagent, 50 ~1 of 50 from Pharmacia Fine Chemicals (Piscataway, PM FMN solution, and 10 ~1 of 100 mM phosN. J.). NADP-agarose (agarose-hexane-nicphate buffer, pH 6.8. The addition of exogeotinamide adenine dinucleotide phosphate, nous BL is not necessary. The light response AGNADP type 4) was from Sigma Chemical was measured with a luminometer 10 s after Company (St. Louis, MO.). All the biolumithe injection, and the 30-s count rate correnescent reagents, Decanal reagent, FMN-0, sponded to the contaminating oxidoreductase NADH, and NADPH, were available from activities. Packard Instrument Company, Inc. (Downers Enzyme puriJication. The initial procedures Grove, Ill.). They were prepared according to in purification of BL from V. harveyi were manufacturers’ direction. Ammonium sulfate similar to the methods reported by Gunsalusused for enzyme work was procured from Re- Miguel et al. (9) and Baldwin et al. (10). The search Plus Laboratories (Denville, N. J.). “reverse-affinity chromatographies” to remove Bacteria. The ingredients of the high salt the D’ase and P’ase were modifications of the medium and the cell culture of the luminous methods described by Jablonski and Debacteria were as described previously by Gun- Luca (8). All steps were carried out at 2-8°C. The salus-Miguel et al. (9). The V. harveyi (M-14) frozen bacterial cells were thawed overnight bacteria cells were grown and harvested at Biological Laboratories, Harvard University. and then lysed until no further increase in BL The frozen bacterial paste was then trans- activity was observed. The enzyme was then absorbed to the DEAE-cellulose, stepwise ported back to our laboratory. Protein assay. Protein was determined by eluted with 50-500 mM phosphate buffer (pH absorbance at 280 nm (A& spectrophoto6.8, containing 100 pM DTT), and precipitated metrically with a uv monitor (ISCO Type 6 at 70% ammonium sulfate. The pellet was reoptical unit, Lincoln, Neb.) connected to the suspended in a minimal amount of 50 mM buffer and dialyzed thoroughly in the same effluent of the column or was determined chemically according to the method of Lowry buffer before applying it to the following et al. (11). DEAE-Sephadex A-50 (Fig. 1) and Sephadex Enzyme assay. BL activity was measured G- 100 column. The fractions containing major BL activity were pooled from the column with photochemically reduced flavin, FMNH;! (12,13). The 20 ~1 of purified enzyme was effluent and concentrated as described above. mixed with 30 ~1 of assay solution (1:500 of At this point, the BL enzyme preparation was Decanal reagent: 100 mM phosphate buffer, ready to undergo final purification steps inpH 6.8). Then 50 ~1 of FMNH2 (50 pM) was volving affinity chromatography. injected at room temperature to initiate the The contaminating D(P)‘ase activity in the bioluminescence reaction ([b]). Light response BL fraction was further removed by two difwas integrated for 30 s on a Packard Model ferent approaches. The first procedure was to 6 100 Picolite (luminometer from Packard In- go through two consecutive reverse-affinity strument Co., Inc.). The light intensity ex- chromatographies, i.e., the NADP agarose to pressed in counts/s is proportional to the lu- retain P’ase and then 5’-AMP-Sepharose 4B to adsorb D’ase, and leaving BL free of the ciferase concentration. The contaminating two reductases in the effluent. The NADP specific D( P)‘ase and nonspecific oxidoreductase (reductases use NADH or NADPH as agarose column has high affinity for enzymes

CHROMATOGRAPHIC

PURIFICATION

OF BACTERIAL

LUCIFERASE

139

BL

1;o

200 I

300 I

400 1

I

500 Fractions

1

600

(ml)

FIG. 1. DEAE-Sephadex A-50 column chromatography of a partially purified BL extracted from Vibrio (Beneckea) harveyi. The resuspended and dialyzed pellet (see Materials and Methods) after DEAE-Celhdose elution was applied onto a 2.5 cm X 53-cm column equilibrated with the resuspension buffer. The 10 ml fractions were eluted with a linear gradient of 100 to 700 mM phosphate buffer (100 PM DTT, pH 6.8), and assayed for absorbance at 280 nm (-), for BL (0 -Cl), and for oxidoreductase activity with NADH (0 - 0) and NADPH (0 - 0).

requiring NADP+ as a cofactor and the enzyme can only be eluted with NADP+ containing buffers. The S-AMP-Sepharose 4B column, possessing strong binding for NAD+dependent dehydrogenases, was operated with the same principle as the “reverse” NADP agarose chromatography. Aliquots of the pooled fractions resulted from two consecutive reverse-affinity chromatographies were assayed for all three enzyme activities. The second method was to pass the contaminating BL fraction through a single type of affinity chromatography. The Blue Sepharose CL-6B has affinity for enzymes requiring adenylyl containing cofactors (including NADf and NADP+), and thereby can retain both D’ase and P’ase on the same column. Four grams of the freeze-dried Blue Sepharose CL-6B gel powder was prepared in a column onto which the purified BL fraction from Sephadex G- 100 was applied.

RESULTS AND DISCUSSION

Initially, most of the D(P)‘ase activities were separated from the BL activity by batchwise elution of the crude extract of I’. harveyi with DEAE-cellulose and by ammonium sulfate fractionation. The remaining reductase activities were resolved into two overlapping peaks, D’ase and P’ase, on a DEAE-Sephadex A-50 chromatogram (Fig. 1). The isolated BL was then subjected to the final steps of removing the reductase activities. These purification schemes and the results obtained are typical of and similar to those reported by other investigators (6,7,9), and therefore are not described in detail in this paper. We would like, however, to emphasize the final purification steps aiming toward the removing of the residual trace amount of D(P)‘ase activities tightly associated with the BL. Various techniques were tried in order to

140

TENLIN TABLE

S. TSAI

1

unsuccessful trials in our laboratory include butyl-agarose, hexyl-agarose, octyl-agarose, amino-hexyl-Sepharose 4B, and chromatofocusing using PBE 94 and polybuffer 74 from Pharmacia. The D’ase and P’ase from V. harveyi have been purified to homogeneity using S-AMPSepharose 4B and NADP-agarose affinity chromatography, respectively, as the final step of purification (8). During the purification of BL, the affinity gels were used in the “reverse” sense that the contaminating reductases are strongly bound to the gels. The BL, unadsorbing to the gel, will void in the first effluent. The reductases are left behind binding to the column material which can later be eluted with increasing concentrations of NAD+ or NADP+. When BL was initially purified

REMOVAL OF OXIDOREDUCTASES FROM BACTERIAL LUCIFERASE BY BLUE SEPHAROSE CLdB AF’FINITY CHROMATOGRAPHY

Specific activity (counts/s/rg protein) Enzymes BL D’ase P’ase

Before column

After column

Removal” @)

x lo5 0.16 x lo* 0.21 x 102

85

X lo5 1.09 x 102 1.71 x 102

2.78

2.67

4 88

’ (Activity before column - activity after column)/activity before column, X 100.

recover the maximal BL activity and to eliminate impurities, especially specific (D’ase, P’ase) and nonspecific oxidoreductases. These

so so

a. .Z .? ii

d 0 x

80 80

70

5

E ‘ii

60

2 s

50

s

40

0

30 0

i 20

10

0

5

10

Fraction

20

15

25

30

100 50 0

Number

2. Separation of D’ase and P’ase activity from BL by Blue Sepharose CL-6B. BL (9 mg protein/ml) was applied onto the 0.7 X 20-cm column equilibrated with 100 mM phosphate buffer (100 pM DTT, pH 6.8). Fractions (3 ml) were collected with the equilibrating buffer at a flow rate of 2 ml/l0 min. The fractions were assayed for all three enzyme activities: BL (O), D’ase (O), and P’ase (0). FIG.

CHROMATOGRAPHIC

PURIFICATION

TABLE 2 COMPARISON OF BLUE SEPHAROSE CLdB TO Two SEQUENTIAL REVERSE-AFFINITY CHROMATOGRAPHIES IN REMOVING OXIDOREDUCTASES

Specific activity (counts/s/fig protein) Enzymes BL D’ase P’ase

Blue Sepharose CLdB 2.49 0.69 0.84

x lo5 X 10’ x lo*

Two reverse-affinity chromatographies” 3.19 x 105 x IO* 1.10 x 102

0.60

’ NADP agarose, and then 5’-AMP Sepharose 4B.

OF BACTERIAL

LUCIFERASE

141

step affinity chromatography are compared with those obtained from two affinity columns in Table 2. The results indicated that Blue Sepharose CL-6B can replace the two consecutive reverse-affinity chromatographies in removing the last contaminating specific and/ or nonspecific reductases from BL. Bacterial luciferase devoid of associating D(P)‘ase can be purified with a simple one-step affinity chromatography. This extra pure BL preparation not only offers the possibility of a highquality luminescent reagent for many uncoupled or coupled bioluminescent reactions, but also is a prerequisite for employing it in bioluminescent enzyme-linked immunoassays.

through NADP agarose, only 15% of the contaminating reductase activities was bound to REFERENCES the gel. The remaining impurities were still 1. Hastings, J. W. (1968) Annu. Rev. Biochem. 37,597significant. The enzyme had to be processed 630. through a second affinity chromatography (S2. McCapra, F., and Hysert, D. W. (1973) Biochem. AMP-Sepharose 4B) to exclude residual reBiophys. Res. Commun. 52,298-304. 3. Dunn, D. K., Michaliszyn, G. A., Bogacki, I. G., and ductase activities. It is time consuming and Me&hen, E. C. (1973) Biochemistry 12,491 l-4918. not economical to perform two sequential af4. Strehler, B. L., and Cormier. M. J. (1954) Arch. finity-chromatographic procedures. Therefore, Biochem. Biophys. 53, 138- 156. seeking other means to obtain a homogeneous 5. Duane, W. C., and Hastings, J. W. (1975) Mol. Cell. BL which contains a minimal amount of reBiochem. 6, 53-64. 6. Gerlo, E., and Charlier, J. (1975) Eur. J. Biochem. ductases is the purpose of this study. 57,46 l-467. Results shown in Table 1 indicated that I. Michaliszyn, G. A., Wing, S. S., and Meighen, E. A. Blue Sepharose CLdB is very effective in re(1977) J. Biol. Chem. 252,7495-1499. moving D’ase and P’ase and protecting BL ac8. Jablonski, E., and DeLuca, M. (1977) Biochemistry tivity intact. The specific activity of all three 16,2932-2936. 9. Gunsalus-Miguel, A., Meighen, E. A., Nicoli, M. Z., enzymes was tabulated before and after the Nealson, K. H., and Hastings, J. W. (1972) J. Biol. column. Eighty-five percent of the D’ase and Chem. 247,398-404. 88% of the P’ase activity were removed, with 10. Baldwin, T. O., Nicoli, M. Z., Becvar, J. E., and Hastrecovery of 96% of the BL activity, all accomings, J. W. (1975) J. Biol. Chem. 250,2763-2168. plished in a single step of chromatographic Il. Lowry. 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. L. (1954) J. Biol. Chem. 193, 265procedure. This method is simple and fast and results in a BL preparation containing less than 12. 275. Nickerson, W. J., and Strauss, G. (1960) J. Amer. 0.0 1% reductase activities. The chromatogram Chem. Sot. 82,5007-5008. of the Blue Sepharose CL-4B is depicted in 13. Strauss, G., and Nickerson, W. J. (1961) J. Amer. Fig. 2 and the results obtained from the oneChem. Sot. 83,3 187-3 I9 1.