JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 6 131-136 (1991)
Bovine Serum Albumin Interacts with Bacterial Luciferase John C. Makernson* and J. Woodland H a s t i n g s Department of Biological Sciences, Florida International University, University Park, Miami, FL 331 99, USA and Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138, USA
Bovine serum albumin (BSA) affects the amount o f light obtained f r o m bacterial luciferase by competing w i t h luciferase f o r one o f the luciferase substrates, the aldehyde. A t l o w aldehyde concentrations BSA behaves as an inhibitor, b u t a t high aldehyde concentrations BSA relieves substrate inhibition. BSA reversibly binds decanal w i t h a Ksi= 3.36kmol/l. approximately half the affinity o f luciferase f o r decanal (K, = 1.5 pmol/l). BSA also increased the rate o f intermediate II dark decay. The data suggest t h a t this involves a direct protein-protein (BSA-luciferase) interaction. Keywords:
Luciferase; bovine serum albumin
INTRO DUCTlON
Bovine serum albumin (BSA) is normally added to the standard assay buffer for bacterial luciferase because a greater initial intensity is obtained in the single injection (FMNH, injection) assay (Hastings et al., 1978). This has been explained as a ‘buffering out’ or ‘protection from’ the inhibitory effect of high concentrations of one of the substrates of the reaction, the aldehyde (Baumstark e l al., 1979; Holzman and Baldwin, 1983), due to the reversible binding of aldehyde to BSA. Further, BSA stimulates a reaction between cyanide and aldehyde to form an inhibitor complex of bacterial luciferase (Makemson, 1990). We have discovered that BSA has other effects on luciferase. We show here that BSA affects both the rate at which I,,, or I , is achieved in the single injection assay and the rate at which the luciferase
flavin hydroperoxide intermediate (11) decays. Additionally, BSA helps to eliminate the adverse effects of short chain alcohols on luciferase. These effects may be similar to the protective and other unexplained effects of BSA on firefly luciferase (Carrico et al., 1978) which result in increased light production.
MATERIALS A N D METHODS Enzyme
Bacterial luciferase was purified from Vibrio harueyi strain B-392 by the method of Hastings et al. (1978) and Holzman and Baldwin (1982) utilizing ammonium sulphate fractionation, DEAE cellulose fractionation (batch) and affinity chromatography
Abbreviations: BSA, bovine serum albumin; FMNH,, reduced flavin mononucleotide; DEAE, diethylaminoethyl; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; I , , initial intensity. *Author for correspondence.
0884-3996/91/0200131 06$05.00 0 1991 by John Wiley & Sons, Ltd.
Received 5 September 1990 Revised I 2 December 1990
132
J. C. MAKEMSON AND J. W. HASTINGS
on 2,2-diphenylpropylamine-Sepharose. SDSPAGE gels of the purified preparation possessed the typical alpha and beta bands corresponding to 40 kDa and 38 kDa of bacterial luciferase and two other minor bands (24 kDa and < 10 kDa) which represented less than 15% of the protein in the purified preparation. Luciferase was measured by the standard assay using catalytically reduced FMNH, (0.05 mmol/ 1, 1 ml) to initiate the reaction with the other ingredients in 1 ml of 0.05 mol/l phosphate buffer, pH 7.0 with (or without, see below) 0.2% BSA with 25 pmol/l decanal (Hastings et al., 1978) at 25°C. Decanal was dissolved in ethanol (9.4 pl decanal in 10 ml ethanol) to make a 5 mmol/l stock solution; 5 pl was added to the assay mixture. The maximal initial intensity (I,) was recorded using a photomultiplier photometer (Mitchell and Hastings, 1971) using the light standard of Hastings and Weber (1 963). Intermediate II decay
The decay of intermediate I1 was measured in 0.05 mol/l phosphate buffer, pH 7.0, at 25°C. Microlitre amounts of enzyme were added to 1.0 ml of buffer (i inhibitor). One ml of 0.05 mmol/l FMNH, was injected at time zero. Just prior to specific time intervals, 50 p1 of the reaction mixture were added to a lumacuvette in an RGM photometer and at specific times 100 p1 ofa 1:5 dilution
of 5 mmol/l decanal in the buffer was injected into the lumacuvette. This concentration of decanal was optimal for producing a Aash of light from the amount of intermediate 11-inhibitor complex. The I , was recorded on a Varian Techtron strip chart recorder and plotted vs time of aldehyde injection to obtain the decay rate of the intermediate 11-inhibitor complex or intermediate II alone. For the decay of intermediate I1 alone and for experiments in which the decay was rapid, samples were taken at 20-second intervals and no fewer than six samples were used to calculate the decay rate. The decay rate was calculated using a least squares linear regression of the natural log of I , vs time. The regression coefficients (r) for all data presented were greater than 0.91. RESULTS AND DISCUSSION
In the standard assay, BSA affected the I, depending upon the amount of aldehyde present (Baumstark et al., 1979; Fig. 1). In this experiment, the aldehyde, decanal, was sonicated into an emulsion in distilled water to exclude the effect of ethanol (Hastings et al., 1966). Fig. 1 shows that, without BSA, there was less light (I,-max) emitted but the amount of aldehyde needed to produce I,,, was less: with no BSA I,-max is at 7.5 pmol/l decanal and with 0.1 % and 0.2% BSA I,-max is at 25 pmol/l while it is 36 pmol/l for 0.4% BSA and 75 pmol/l for 0.6% BSA.
. . .
1200 1000 v,
800
'=
Z 3
BSA
600
0.6%
400
0.4%
200
0.2% 0 1%
I
2
2
10
100
1000
[Decanal, p M ]
Figure 1. Effect of bovine serum albumin on the lo of the standard luciferase reaction as a function of aldehyde concentration. (Bovine serum albumin in the standard assay mixture at 0.1% (closed circles), 0.2% (open triangles), 0.4% (closed triangles), 0 6% (open boxes). or none (open circles)
133
BSA AND LUClFERlN
At low concentrations of decanal there should be little involvement of aldehyde as an inhibitor binding the putative, second site ( K , = 15 pmol/l (Holzman and Baldwin, 1983)). At aldehyde concentrations below that needed to achieve I,-max, BSA appears to act as an inhibitor. Another effect of BSA concentration in this assay procedure is the rate at which the I, is attained ('turn-on kinetics'). These turn-on rates are really accelerations to I, (in light units) which itself is a rate (which can be expressed in quanta per second). Increasing the concentration of BSA slows down the rate of attainment of I,: in this experiment, the intensity of the reaction was recorded so that each 0.25 s interval could be used to calculate the initial rate of attainment of I, at each concentration of aldehyde for various concentrations of BSA. When this data is plotted as a Lineweaver-Burk plot (Fig. 2), the 'turn on' KM (10 pmol/l, no BSA curve) appears greater than six times the actual K Mfor decanal (1.5 pmol/l; see below and Holzman and Baldwin (1983)). This suggests that aldehyde may have positive effects: accelerating the attainment of I, at concentrations above the actual KM. Although the regression curves d o not intersect the ordinate at exactly the same place, BSA appeared to act as a competitive inhibitor by increasing the apparent KMof the turn-on kinetics. The apparent K, for BSA from these data is 9.4pmol/l. This value, however, does not describe the interaction of BSA with decanal as a competitive inhibitor that removes substrate by binding with it (Webb, 1963).
Assuming that decanal binds BSA reversibly, the affinity of BSA for decanal can be estimated by first calculating the concentration of free decanal, [S] which can be estimated from:
Is]
=
[SII(KM)(l
-
i)/(KM + i[stl)
where [S,] is the total concentration of decanal, KM is the Michaelis constant, and i is the fractional amount of inhibition (Webb, 1963). Then [S] can be used to calculate the interaction of BSA with decanal, Ksi (Webb, 1963) from:
This calculation ignores the possibility that BSA may have more than one binding site for aldehyde. The K,, for BSA and decanal using the data from Fig. 2 comes to 3.36 pmol/l (k0.97, n = 16). At high aldehyde concentrations, the inhibitory effect of these saturating amounts of decanal was partly removed with an increase in the concentration of BSA (Fig. 1). This suggests that BSA binds rapidly and reversibly with aldehyde so that an additional molecule of aldehyde does not attach to the second aldehyde (inhibitory) site on luciferase. In the standard assay with BSA short chain alcohols appeared to have no effect: adding absolute ethanol up to 20 pl per assay did not change the I , or the apparent kinetics of decay. It then seemed reasonable, therefore, to use ethanolic solutions of aldehyde rather than water-based emul-
-
01-0.4 -0.2
"
0.0
'
'
0.2
'
0.4
0.6
0.8
1.0
l/pl DECANAL Figure 2. Lineweaver-Burk plot of the effect of bovine serum albumin on the 'turn-on' kinetics of the standard luciferase reaction as a function of aldehyde concentration (Bovine serum albumin in the standard assay mixture at 0.1% (closed circles), 0 2% (open triangles). 0.4% (closed triangles), 0.6% (open boxes), or none (open circles)
J. C. MAKEMSON AND J. W. HASTINGS
Table 1. Effect of short chain alcohols on luciferase standard assay without bovine serum albumin at 25°C Alcohol
Concentration
(mmol/l) None Methanol Ethanol Propanol
139 85 67
Light decay constant ( s - l )
Quantum yield
0.229 0.323 0.278 0.371
755 758 700
71 4
Alcohols added at 10 pl per assay, concentration calculated from the final volume of assay (2ml).
a
sions of aldehyde because the aqueous emulsions begin to slowly separate: they must be made fresh and titrated (to give maximal I,) every couple of hours. Stoppered aldehyde solutions in ethanol were stable over longer periods of time (days). Ethanol and other short chain alcohols, however, do have an effect on luciferase assay without BSA: both the I , (Hastings et al., 1966) and the decay kinetics (Table 1) were increased by the presence of these short chain alcohols resulting in minor changes in the quantum yield. Short chain alcohols (C2 to C4) have also been found to increase dark decay rate of intermediate 11 (data not shown) and this may be related in part to the increase in light decay rate in the standard assay without BSA (Table 1). Although the mechanism by which the short chain alcohols cause the in-
r-----1-
10
05
crease in I , and decay rate is not known, they must interact directly since they have no effect in the presence of BSA (which presumably binds them with a higher affinity than does luciferase). Correcting for the effect of ethanol, LineweaverBurk plots of 1/I, vs l/pl aldehyde in the assay with no BSA present are parabolic and one cannot estimate the aldehyde KM. Holzman and Baldwin (1983) estimated the K M in different buffers from low concentrations of aldehyde taking into account the inhibitory effects of aldehyde and obtained K , values in the range of 1.2 to 1.8 pmol/l with no BSA. With 0.2 % BSA the K,-app was 9.0 pmol/l and this term includes the interference of BSA. The KM for aldehyde can be estimated from Lineweaver-Burk plots from standard assay data using different concentrations of BSA and using data points from the smallest concentrations of aldehyde (those below that needed to produce I,-max). Using the data from FIg. 1, the Lineweaver-Burk plot (Fig. 3) shows that the BSA-containing assays produced a cluster of regression curves intersecting at the same point (0.998,0.992, 1.09,1.05 for 0.1 %, 0.2%, 0.4% and 0.6% BSA respectively) that were well separated from the curve based on assays without BSA. The curve without BSA most likely contains aldehyde inhibition (shifted upward), thus to calculate the KM from this data one can use only the data in the presence of BSA in a region where it is acting like a classical competitive inhibitor. Thus, standard Michaelis-Menten competitive inhibition kinetic
r-----1 '
0.0
05
'
'
'
'
1 .o
l/pl 3 e c o n o l Figure 3. Lineweaver-Burk plot of the effect of bovine serum albumin on the standard luciferase reaction as a function of aldehyde concentration. (Data plotted from Fig. 1 . bovine serum albumin in the standard assay mixture at 0.1% (closed circles). 0 2 % (open triangles), 0.4% (closed triangles). 0.6% (open boxes), or none (open circles)
BSA AND LUClFERlN
135
20
0
60
40
80
100
120
SECONDS Figure 4. Effect of bovine serum albumin on the dark decay of luciferase intermediate I1 with no aldehyde added. (Bovine serum albumin added at 0.02 mg/ml (closed circles), 0.05 mg/rnl (open triangles), 0.1 mg/ml (closed triangles), 0.2 rng/ml (open boxes), 0.4 mg/ml (closed boxes), or none (open circles)
analysis can be used. Using simultaneous equations for the solution of K, (which algebraically removes KM) from the six combinations of BSA concentrations, the Ki of BSA in this analysis is 1.88 ( & 0.65) x lop5mol/l. Using this K , one can go back and calculate the KM from the data using the four concentrations of BSA, and the KM (aldehyde binding to luciferase) then is 1.51 ( 0.047) pmol/l; this is the same value obtained by Holzman and Baldwin (1983).
In another approach, Sobolev and Danilov (1988) have estimated the KM for decanal in the coupled assay (NADH-FMN oxidoreductase/luciferase) to be 60 pmolll; this value may be too high, possibly because the coupled assay has other ingredients which could affect aldehyde availability to luciferase. This is similar to the apparent KM for aldehyde in the presence of BSA being increased from 1.5 pmol/l to 9.0 pmol/l (Holzman and Baldwin, 1983).
*
0.08 0.07 0.06 r
I
Ycn
0.05
J
0.04
0.03
002' 0
'
'
2
'
'
4
'
'
6
'
'
'
8
'
10
'
'
12
'
'
14
'
'
16
'
'
18
'
J
20
[BSA. pM1 Figure 5. Decay rate of luciferase intermediate II as a function of bovine serum albumin concentration. (Initial (linear) rates were used t o calculate the first order decay constant, k, in units of s - ' )
136
J. C. MAKEMSON AND J. W. HASTINGS
REFERENCES In the absence of aldehyde, BSA increased not only the amount of intermediate I1 formed but also the rate of intermediate I1 dark decay (Figs 4 and Baumstark, A., Cline, T. and Hastings, J. W. (1979). Reversible steps in the reaction of aldehydes with luciferase interme5). This effect is specific for BSA, chicken-egg diates. Arch. Biochem. Biophys., 193, 449-455. albumin had no effect on the decay of intermediate Carrico, R. J., Johnson, R. D. and Boguslaski, R. C. (1978). 11. Addition of 0.2 % chicken-egg albumin resulted ATP-labeled ligands and firefly luciferase for monitoring specific protein-binding reactions. Methods Enzymol., 57, in a decay rate of k = 0.027 s-' compared to 113-122. 0.025 s - ' with no chicken-egg albumin at 24°C. Hastings. J. W. and Weber, G . (1963). Total quantum flux of This difference is not significant when compared to isotropic sources. J . Opt. Soc. Am., 53, 1410-1415. the addition of 0.2% BSA which produced a dra- Hastings, J. W., Gibson, Q. H., Friedland, J. and Spudich, J. (1 966). Molecular mechanisms in bacterial bioluminescence: matically higher decay rate: k = 0.071 s-'. on energy storage intermediates and the role of aldehyde in The increased dark decay rate with BSA showed the reaction. In Bioluminescence in Progress, Johnson, F. H. saturation (Fig. 5) and could result from a direct and Haneda, Y. (Eds), Princeton University Press, Princeinteraction between BSA and luciferase intermeton, NJ, pp. 151-186. diate I1 by effecting either a conformational change Hastings, J . W., Baldwin, T. 0.and Nicoli, M. 2. (1978). Bacterial luciferase: assay, purification and properties. Methods in luciferase that accelerates the release of the flavin Enzymology, 57, 135-152. hydroperoxide or by directly binding and removing Holzman, T. F. and Baldwin, T. 0.(1982). Isolation of bacterial the tightly bound flavin hydroperoxide from luciluciferases by affinity chromatography on 2,2-diphenylproferase. In either case, these data suggest that BSA pylamine-Sepharose: phosphate-mediated binding to an immobilized substrate analogue. Biochemistry, 21, 6194-6201. physically interacts with luciferase to accelerate Holzman, T. F. and Baldwin, T. 0.(1983). Reversible inhibition dark decay. of bacterial luciferase catalyzed bioluminescent reaction by BSA appears to affect luciferase assay in several aldehyde substrate: kinetic mechanism and ligand effects. ways: as a competitive inhibitor reversibly removBiochemistry, 22, 2838-2846. ing aldehyde from solution; binding small molecu- Makemson, J. C. 1990. A cyanide-aldehyde complex inhibits bacterial luciferase. J . Bacteriol., 172,4725-4727. lar weight alcohols thereby eliminating their effects; and interacting directly with luciferase in some Makemson, J. C. and Hastings, J. W. (1979). Inhibition of bacterial luciferase by pargyline. Arch. Biochem. Biophys., manner to influence the rate of intermediate I1 196, 396-402. decay through the dark pathway. Mitchell, G. and Hastings, J. W. (1971). A stable, inexpensive Ac k now I edgeme nt T h a n k s a r e d u e t o L a u r i e Richardson for critical review of t h e manuscript.
solid state photomultiplier photometer. Anal. Biochem., 39, 243-250. Sobolev, A. Yu. and Danilov, V. S. (1988). Influence of decylamine on the reaction of bacterial luciferase. Biokhimiya (English translation), 53, 788-793. Webb, J. L. (1963). Enzyme and Metabolic Inhibitors, Vol I, Academic Press, NY.
Bovine Serum Albumin Interacts with Bacterial Luciferase John C. Makernson* and J. Woodland H a s t i n g s Department of Biological Sciences, Florida International University, University Park, Miami, FL 331 99, USA and Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138, USA
Bovine serum albumin (BSA) affects the amount o f light obtained f r o m bacterial luciferase by competing w i t h luciferase f o r one o f the luciferase substrates, the aldehyde. A t l o w aldehyde concentrations BSA behaves as an inhibitor, b u t a t high aldehyde concentrations BSA relieves substrate inhibition. BSA reversibly binds decanal w i t h a Ksi= 3.36kmol/l. approximately half the affinity o f luciferase f o r decanal (K, = 1.5 pmol/l). BSA also increased the rate o f intermediate II dark decay. The data suggest t h a t this involves a direct protein-protein (BSA-luciferase) interaction. Keywords:
Luciferase; bovine serum albumin
INTRO DUCTlON
Bovine serum albumin (BSA) is normally added to the standard assay buffer for bacterial luciferase because a greater initial intensity is obtained in the single injection (FMNH, injection) assay (Hastings et al., 1978). This has been explained as a ‘buffering out’ or ‘protection from’ the inhibitory effect of high concentrations of one of the substrates of the reaction, the aldehyde (Baumstark e l al., 1979; Holzman and Baldwin, 1983), due to the reversible binding of aldehyde to BSA. Further, BSA stimulates a reaction between cyanide and aldehyde to form an inhibitor complex of bacterial luciferase (Makemson, 1990). We have discovered that BSA has other effects on luciferase. We show here that BSA affects both the rate at which I,,, or I , is achieved in the single injection assay and the rate at which the luciferase
flavin hydroperoxide intermediate (11) decays. Additionally, BSA helps to eliminate the adverse effects of short chain alcohols on luciferase. These effects may be similar to the protective and other unexplained effects of BSA on firefly luciferase (Carrico et al., 1978) which result in increased light production.
MATERIALS A N D METHODS Enzyme
Bacterial luciferase was purified from Vibrio harueyi strain B-392 by the method of Hastings et al. (1978) and Holzman and Baldwin (1982) utilizing ammonium sulphate fractionation, DEAE cellulose fractionation (batch) and affinity chromatography
Abbreviations: BSA, bovine serum albumin; FMNH,, reduced flavin mononucleotide; DEAE, diethylaminoethyl; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; I , , initial intensity. *Author for correspondence.
0884-3996/91/0200131 06$05.00 0 1991 by John Wiley & Sons, Ltd.
Received 5 September 1990 Revised I 2 December 1990
132
J. C. MAKEMSON AND J. W. HASTINGS
on 2,2-diphenylpropylamine-Sepharose. SDSPAGE gels of the purified preparation possessed the typical alpha and beta bands corresponding to 40 kDa and 38 kDa of bacterial luciferase and two other minor bands (24 kDa and < 10 kDa) which represented less than 15% of the protein in the purified preparation. Luciferase was measured by the standard assay using catalytically reduced FMNH, (0.05 mmol/ 1, 1 ml) to initiate the reaction with the other ingredients in 1 ml of 0.05 mol/l phosphate buffer, pH 7.0 with (or without, see below) 0.2% BSA with 25 pmol/l decanal (Hastings et al., 1978) at 25°C. Decanal was dissolved in ethanol (9.4 pl decanal in 10 ml ethanol) to make a 5 mmol/l stock solution; 5 pl was added to the assay mixture. The maximal initial intensity (I,) was recorded using a photomultiplier photometer (Mitchell and Hastings, 1971) using the light standard of Hastings and Weber (1 963). Intermediate II decay
The decay of intermediate I1 was measured in 0.05 mol/l phosphate buffer, pH 7.0, at 25°C. Microlitre amounts of enzyme were added to 1.0 ml of buffer (i inhibitor). One ml of 0.05 mmol/l FMNH, was injected at time zero. Just prior to specific time intervals, 50 p1 of the reaction mixture were added to a lumacuvette in an RGM photometer and at specific times 100 p1 ofa 1:5 dilution
of 5 mmol/l decanal in the buffer was injected into the lumacuvette. This concentration of decanal was optimal for producing a Aash of light from the amount of intermediate 11-inhibitor complex. The I , was recorded on a Varian Techtron strip chart recorder and plotted vs time of aldehyde injection to obtain the decay rate of the intermediate 11-inhibitor complex or intermediate II alone. For the decay of intermediate I1 alone and for experiments in which the decay was rapid, samples were taken at 20-second intervals and no fewer than six samples were used to calculate the decay rate. The decay rate was calculated using a least squares linear regression of the natural log of I , vs time. The regression coefficients (r) for all data presented were greater than 0.91. RESULTS AND DISCUSSION
In the standard assay, BSA affected the I, depending upon the amount of aldehyde present (Baumstark et al., 1979; Fig. 1). In this experiment, the aldehyde, decanal, was sonicated into an emulsion in distilled water to exclude the effect of ethanol (Hastings et al., 1966). Fig. 1 shows that, without BSA, there was less light (I,-max) emitted but the amount of aldehyde needed to produce I,,, was less: with no BSA I,-max is at 7.5 pmol/l decanal and with 0.1 % and 0.2% BSA I,-max is at 25 pmol/l while it is 36 pmol/l for 0.4% BSA and 75 pmol/l for 0.6% BSA.
. . .
1200 1000 v,
800
'=
Z 3
BSA
600
0.6%
400
0.4%
200
0.2% 0 1%
I
2
2
10
100
1000
[Decanal, p M ]
Figure 1. Effect of bovine serum albumin on the lo of the standard luciferase reaction as a function of aldehyde concentration. (Bovine serum albumin in the standard assay mixture at 0.1% (closed circles), 0.2% (open triangles), 0.4% (closed triangles), 0 6% (open boxes). or none (open circles)
133
BSA AND LUClFERlN
At low concentrations of decanal there should be little involvement of aldehyde as an inhibitor binding the putative, second site ( K , = 15 pmol/l (Holzman and Baldwin, 1983)). At aldehyde concentrations below that needed to achieve I,-max, BSA appears to act as an inhibitor. Another effect of BSA concentration in this assay procedure is the rate at which the I, is attained ('turn-on kinetics'). These turn-on rates are really accelerations to I, (in light units) which itself is a rate (which can be expressed in quanta per second). Increasing the concentration of BSA slows down the rate of attainment of I,: in this experiment, the intensity of the reaction was recorded so that each 0.25 s interval could be used to calculate the initial rate of attainment of I, at each concentration of aldehyde for various concentrations of BSA. When this data is plotted as a Lineweaver-Burk plot (Fig. 2), the 'turn on' KM (10 pmol/l, no BSA curve) appears greater than six times the actual K Mfor decanal (1.5 pmol/l; see below and Holzman and Baldwin (1983)). This suggests that aldehyde may have positive effects: accelerating the attainment of I, at concentrations above the actual KM. Although the regression curves d o not intersect the ordinate at exactly the same place, BSA appeared to act as a competitive inhibitor by increasing the apparent KMof the turn-on kinetics. The apparent K, for BSA from these data is 9.4pmol/l. This value, however, does not describe the interaction of BSA with decanal as a competitive inhibitor that removes substrate by binding with it (Webb, 1963).
Assuming that decanal binds BSA reversibly, the affinity of BSA for decanal can be estimated by first calculating the concentration of free decanal, [S] which can be estimated from:
Is]
=
[SII(KM)(l
-
i)/(KM + i[stl)
where [S,] is the total concentration of decanal, KM is the Michaelis constant, and i is the fractional amount of inhibition (Webb, 1963). Then [S] can be used to calculate the interaction of BSA with decanal, Ksi (Webb, 1963) from:
This calculation ignores the possibility that BSA may have more than one binding site for aldehyde. The K,, for BSA and decanal using the data from Fig. 2 comes to 3.36 pmol/l (k0.97, n = 16). At high aldehyde concentrations, the inhibitory effect of these saturating amounts of decanal was partly removed with an increase in the concentration of BSA (Fig. 1). This suggests that BSA binds rapidly and reversibly with aldehyde so that an additional molecule of aldehyde does not attach to the second aldehyde (inhibitory) site on luciferase. In the standard assay with BSA short chain alcohols appeared to have no effect: adding absolute ethanol up to 20 pl per assay did not change the I , or the apparent kinetics of decay. It then seemed reasonable, therefore, to use ethanolic solutions of aldehyde rather than water-based emul-
-
01-0.4 -0.2
"
0.0
'
'
0.2
'
0.4
0.6
0.8
1.0
l/pl DECANAL Figure 2. Lineweaver-Burk plot of the effect of bovine serum albumin on the 'turn-on' kinetics of the standard luciferase reaction as a function of aldehyde concentration (Bovine serum albumin in the standard assay mixture at 0.1% (closed circles), 0 2% (open triangles). 0.4% (closed triangles), 0.6% (open boxes), or none (open circles)
J. C. MAKEMSON AND J. W. HASTINGS
Table 1. Effect of short chain alcohols on luciferase standard assay without bovine serum albumin at 25°C Alcohol
Concentration
(mmol/l) None Methanol Ethanol Propanol
139 85 67
Light decay constant ( s - l )
Quantum yield
0.229 0.323 0.278 0.371
755 758 700
71 4
Alcohols added at 10 pl per assay, concentration calculated from the final volume of assay (2ml).
a
sions of aldehyde because the aqueous emulsions begin to slowly separate: they must be made fresh and titrated (to give maximal I,) every couple of hours. Stoppered aldehyde solutions in ethanol were stable over longer periods of time (days). Ethanol and other short chain alcohols, however, do have an effect on luciferase assay without BSA: both the I , (Hastings et al., 1966) and the decay kinetics (Table 1) were increased by the presence of these short chain alcohols resulting in minor changes in the quantum yield. Short chain alcohols (C2 to C4) have also been found to increase dark decay rate of intermediate 11 (data not shown) and this may be related in part to the increase in light decay rate in the standard assay without BSA (Table 1). Although the mechanism by which the short chain alcohols cause the in-
r-----1-
10
05
crease in I , and decay rate is not known, they must interact directly since they have no effect in the presence of BSA (which presumably binds them with a higher affinity than does luciferase). Correcting for the effect of ethanol, LineweaverBurk plots of 1/I, vs l/pl aldehyde in the assay with no BSA present are parabolic and one cannot estimate the aldehyde KM. Holzman and Baldwin (1983) estimated the K M in different buffers from low concentrations of aldehyde taking into account the inhibitory effects of aldehyde and obtained K , values in the range of 1.2 to 1.8 pmol/l with no BSA. With 0.2 % BSA the K,-app was 9.0 pmol/l and this term includes the interference of BSA. The KM for aldehyde can be estimated from Lineweaver-Burk plots from standard assay data using different concentrations of BSA and using data points from the smallest concentrations of aldehyde (those below that needed to produce I,-max). Using the data from FIg. 1, the Lineweaver-Burk plot (Fig. 3) shows that the BSA-containing assays produced a cluster of regression curves intersecting at the same point (0.998,0.992, 1.09,1.05 for 0.1 %, 0.2%, 0.4% and 0.6% BSA respectively) that were well separated from the curve based on assays without BSA. The curve without BSA most likely contains aldehyde inhibition (shifted upward), thus to calculate the KM from this data one can use only the data in the presence of BSA in a region where it is acting like a classical competitive inhibitor. Thus, standard Michaelis-Menten competitive inhibition kinetic
r-----1 '
0.0
05
'
'
'
'
1 .o
l/pl 3 e c o n o l Figure 3. Lineweaver-Burk plot of the effect of bovine serum albumin on the standard luciferase reaction as a function of aldehyde concentration. (Data plotted from Fig. 1 . bovine serum albumin in the standard assay mixture at 0.1% (closed circles). 0 2 % (open triangles), 0.4% (closed triangles). 0.6% (open boxes), or none (open circles)
BSA AND LUClFERlN
135
20
0
60
40
80
100
120
SECONDS Figure 4. Effect of bovine serum albumin on the dark decay of luciferase intermediate I1 with no aldehyde added. (Bovine serum albumin added at 0.02 mg/ml (closed circles), 0.05 mg/rnl (open triangles), 0.1 mg/ml (closed triangles), 0.2 rng/ml (open boxes), 0.4 mg/ml (closed boxes), or none (open circles)
analysis can be used. Using simultaneous equations for the solution of K, (which algebraically removes KM) from the six combinations of BSA concentrations, the Ki of BSA in this analysis is 1.88 ( & 0.65) x lop5mol/l. Using this K , one can go back and calculate the KM from the data using the four concentrations of BSA, and the KM (aldehyde binding to luciferase) then is 1.51 ( 0.047) pmol/l; this is the same value obtained by Holzman and Baldwin (1983).
In another approach, Sobolev and Danilov (1988) have estimated the KM for decanal in the coupled assay (NADH-FMN oxidoreductase/luciferase) to be 60 pmolll; this value may be too high, possibly because the coupled assay has other ingredients which could affect aldehyde availability to luciferase. This is similar to the apparent KM for aldehyde in the presence of BSA being increased from 1.5 pmol/l to 9.0 pmol/l (Holzman and Baldwin, 1983).
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[BSA. pM1 Figure 5. Decay rate of luciferase intermediate II as a function of bovine serum albumin concentration. (Initial (linear) rates were used t o calculate the first order decay constant, k, in units of s - ' )
136
J. C. MAKEMSON AND J. W. HASTINGS
REFERENCES In the absence of aldehyde, BSA increased not only the amount of intermediate I1 formed but also the rate of intermediate I1 dark decay (Figs 4 and Baumstark, A., Cline, T. and Hastings, J. W. (1979). Reversible steps in the reaction of aldehydes with luciferase interme5). This effect is specific for BSA, chicken-egg diates. Arch. Biochem. Biophys., 193, 449-455. albumin had no effect on the decay of intermediate Carrico, R. J., Johnson, R. D. and Boguslaski, R. C. (1978). 11. Addition of 0.2 % chicken-egg albumin resulted ATP-labeled ligands and firefly luciferase for monitoring specific protein-binding reactions. Methods Enzymol., 57, in a decay rate of k = 0.027 s-' compared to 113-122. 0.025 s - ' with no chicken-egg albumin at 24°C. Hastings. J. W. and Weber, G . (1963). Total quantum flux of This difference is not significant when compared to isotropic sources. J . Opt. Soc. Am., 53, 1410-1415. the addition of 0.2% BSA which produced a dra- Hastings, J. W., Gibson, Q. H., Friedland, J. and Spudich, J. (1 966). Molecular mechanisms in bacterial bioluminescence: matically higher decay rate: k = 0.071 s-'. on energy storage intermediates and the role of aldehyde in The increased dark decay rate with BSA showed the reaction. In Bioluminescence in Progress, Johnson, F. H. saturation (Fig. 5) and could result from a direct and Haneda, Y. (Eds), Princeton University Press, Princeinteraction between BSA and luciferase intermeton, NJ, pp. 151-186. diate I1 by effecting either a conformational change Hastings, J . W., Baldwin, T. 0.and Nicoli, M. 2. (1978). Bacterial luciferase: assay, purification and properties. Methods in luciferase that accelerates the release of the flavin Enzymology, 57, 135-152. hydroperoxide or by directly binding and removing Holzman, T. F. and Baldwin, T. 0.(1982). Isolation of bacterial the tightly bound flavin hydroperoxide from luciluciferases by affinity chromatography on 2,2-diphenylproferase. In either case, these data suggest that BSA pylamine-Sepharose: phosphate-mediated binding to an immobilized substrate analogue. Biochemistry, 21, 6194-6201. physically interacts with luciferase to accelerate Holzman, T. F. and Baldwin, T. 0.(1983). Reversible inhibition dark decay. of bacterial luciferase catalyzed bioluminescent reaction by BSA appears to affect luciferase assay in several aldehyde substrate: kinetic mechanism and ligand effects. ways: as a competitive inhibitor reversibly removBiochemistry, 22, 2838-2846. ing aldehyde from solution; binding small molecu- Makemson, J. C. 1990. A cyanide-aldehyde complex inhibits bacterial luciferase. J . Bacteriol., 172,4725-4727. lar weight alcohols thereby eliminating their effects; and interacting directly with luciferase in some Makemson, J. C. and Hastings, J. W. (1979). Inhibition of bacterial luciferase by pargyline. Arch. Biochem. Biophys., manner to influence the rate of intermediate I1 196, 396-402. decay through the dark pathway. Mitchell, G. and Hastings, J. W. (1971). A stable, inexpensive Ac k now I edgeme nt T h a n k s a r e d u e t o L a u r i e Richardson for critical review of t h e manuscript.
solid state photomultiplier photometer. Anal. Biochem., 39, 243-250. Sobolev, A. Yu. and Danilov, V. S. (1988). Influence of decylamine on the reaction of bacterial luciferase. Biokhimiya (English translation), 53, 788-793. Webb, J. L. (1963). Enzyme and Metabolic Inhibitors, Vol I, Academic Press, NY.
