Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative

Journal of Biochemtcal and Biophysical Methods, 11 (1985) 45-57

45

Elsevier BBM 00471

Real-time fluorescence polarization measurements" interaction of phosphotipase A 2 with a fluorescent lecithin derivative John A. Monti, Michael R. McBride, Steven A. Barker, Janet L. Linton and Samuel T. Christian Neurosciences Program and the Department of Psychiatry, Universzty of Alabama ~n Blrrmngham, Birmingham, AL 35294, U.S.A.

(Received 5 October 1984) (Accepted 10 January 1985)

SummaD~ We have modified an SLM 4800 fluorometer to allow continuous measurement of fluorescence polarization. The modifications included construction of simple mechanical and electronic accessones which allowed polarizer position to be program-controlled by an HP 9815 calculator. With these modifications we were able to follow the interaction of the fluorescent lipid analog 1-acyl-2-(N-4-mtrobenzo-2-oxa-l,3-diazole)-aminododecanoyl phosphatidylcholine (I) with Naja-naja venom phospholipase A 2 (PLA2) (E.C. 3.1.1.4). Upon addition of aliquots of PLA 2 containing up to 85 ng protein to a cuvette containing 1-2 /~M I, the total measured polarization decreased linearly with time for at least 1000 s. Concomitant analyses of equivalent incubation mixtures and analyses of the contents of the cuvette after incubation and collection of fluorescence data revealed time-dependent formation of N-4-nitrobenzo-2oxa-l,3-diazole-aminododecanoic acid (II). The decrease in total measured polarization was accelerated by Ca 2+ and inhibited by EDTA. These data saggest that PLA 2 activity in Naja-naja venom can be measured rapidly at low concentrations of both enzyme (0.01/~g protein) and substrate (1/~M). Since this technique can be used to collect polarization data over time intervaIs as short as 4 s, it should be possible to measure the early changes in polarization during the interaction of fluorescent probes with proteins or membranes, Key words: fluorescence polarization; phospholipase A 2.

Intr, >~'~~ction Fluorescence polarization has been used for many years to investigate protein and membrane dynamics [1-4]. Despite significant advances in fluorescence instrumentation in terms of optics and electronics, measurement of fluorescence polarization has remained essentially a manual procedure requiring orientation and re-orientation 0165-022X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Divismn)

46 of polarizers. As far as we are aware, the first description of an instrument specifically designed and constructed for automated measurement of fluorescence polarization was made by Weber and Bablouzian [5]. This instrument was designed primarily to a11ow measurement of polarization spectra, i.e. the change in polarization as a function of the exciting wavelength. Since then, there have been several reports on automated polarization fluorometers, usually in reference to immunoassay procedures. Spencer et al. [61 described an automated flow-cell polarization fluorometer which utilized a ratio digital voltmeter and a ratio recorder for data collection. Several additional improvements in instrumentation and data collection have been reported by Kelly et al. [7], Jolley et al. [81, and Popelka et al. [9]. As mentioned above, fluorescence polarization and automated instruments for its measurement have been applied to various immunoassay procedures. In these measurements, the fluorochrome is usually bound to a relatively large protein in order to maximize the difference in polarization between the 'free' and ~bound' forms of the labeled ligand. For example, Spencer et al. [6] used FITC-insulin and antiserum in a polarization immunoassay [or porcine insulin. Increasing amounts of added unlabeled insulin competed for the antibody binding sites, displacing the labeled insulin, with an observed decrease influorescence polarization from 0.15 to approximately 0.02. We considered the possibility that this technique might also be applicable to enzyme assays where a fluorescently labeled substrate might be expected to show changes in fluorescence polarization. Over the past few years, this laboratory has reported on the preparation, properties, and applications of fluorescent derivatives of phosphatidylcholine [10,11] and phosphatidylethanolamine [12]. In this communication, we describe relatively simple electronic and mechanical modifications made on an SLM 4800 fluorometer which allow continuous measurement of fluorescence polarization, and present data on the use of this technique for following the interaction of phospholipase A 2 (PLA2) (E.C.3.1.1.4) with a fluorescent derivative of phosphatidylcholine. Measurements of phospholipase activity have been reported which utilize titrimetry [13], radiolabeled phospholipids [14], spectrophotometfic and fluorometric techniques [15,16], and recently, fluorescence polarization, as described by Wolf et al. [17]. These latter investigators used 2-parinaroyl lecithin to measure PLA 2 activity in Crotalus adamanteus venom in a n 'albumin-rich' (10 mg/ml) medium. This procedure requires the incorporation of parinaroyllecithin in egg lecithin vesicles, careful deoxygenatioa of buffers, addition of an antioxidant (butylated hydroxytoluene) to avoid oxidation of parinaric acid and corrections of the measured polarized intensities (Ill and I .L), for both light scattering due to the high concentration of albumin and instrumental anisotropy. We wish to report a method for measuring PLA 2 activity in Naja-naja venom based on real-time continuous polarization measurements. This method for measuring PLA 2 activity uses 1-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)-aminododecanoyl phosphatidylcholine (I) (see Fig. 1), which does not require addition of other lipids, requires no special precautions against oxidation of the fluorochrome, and requires n o corrections for protein-induced or monochromator-induced light

47 0 H

o

,,

I II

//

L

\

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.

|,&,

H C - O - P - O ( C H 2 ) 2 - N ' - Me H

O"

t Me

, - a c y l - 2 - ( N - 4 - nitrobenzo- 2 - oxa- 1,3-d iazole ) GO-a mlnododecanoy I phosphat idylcholine

0 02N

N - (C H2),,- C - O H N

-0 z

N

N - 4 - n itrobenzo -2_- oxe - 1,3- diazole G) - ommododecanoic acid Fig. 1. Structure of

1-acyl-2-(N-4-mtrobenzo-2-o×a-l,3-diazo]e)-amino

dodecanoy] phosphatidylcho]ine

(I) and nitrobenzdiazole-~o-aminododecanoicacid (II).

scattering. This method is capable of detecting less than 0.1 nmol of substrate hydrolyzed per rain. Over a 1000 s interval the observed change in polarization varies linearly with the increased formation of N-4-nitrobenzo-2-oxa-l,3-diazoleaminododecanoic acid (II). Thus, activity can be calculated from the change in polarization. In addition, the real-time, continuous polarization method allows collection of a large number of data points (1 measurement/5 s) immediately following addition of enzyme. Thus, it would appear that this method might be suitable for performing kinetic studies on PLA 2.

Materials

and Methods

The fluorescent phosphatidylcholine derivative (I) used in these studies was a gift from Dr. Walter Shaw, Avanti Polar Lipids, Inc., Birmingham, AL. Solvents were either spectroscopic or HPLC-grade, and were obtained from Burdick and Jackson Laboratories, Muskegon, MI, and Fisher Scientific Co., Pittsburgh, PA. 7-Chloro-4nitrobenzo-2-oxa-l,3-diazole (NBD-C1) was obtained from Aldrich Chemical Co., Inc., Milwaukee, WI. Phospholipase A 2 (Naja-naja venom) was obtained from Sigma and was used without further purification.

Synthesis of H NBD-labeled 12-aminododecanoic acid (ADA) was prepared using a modification of the procedure described by Fager et al. [18] for preparing NBD-labeled

48 a-amino acids, tnto a 3-neck, round bottom flask was added 0.512 g (2.38 mmol) of ADA in 30 ml methanol and 1.0 ml of methanol saturated with sodium acetate. The mixture was refluxed under N 2 with stirring and 0.25 g (1.25 mmol) NBD-C1 in 20 ml methanol was added dropwise over a 10 rain interval. The mixture was refluxed for 30 min after the addition of the NBD-C1 and filtered. The filtrate was evaporated to dryness and the residue was washed with 80 mi of warm benzene/glacial acetic acid (80 : 1, v/v). Material insoluble in b e n z e n e / H O A c was removed by fiRration, and the filtrate was placed in the refrigerator (5°C) overnight. The precipitate which formed was removed by filtration and the filtrate was evaporated to dryness. The residue was washed with approximately 100 ml of acetone and acetone-insoluble material was removed by filtration. Evaporation of the filtrate yielded an oil which, when placed under vacuum, yielded 0.3 g (64% of theoretical) of a dark reddish-brown solid; this final product was stored at 0°C in a dark container.

Spectra Ultraviolet-visible spectra for II were recorded using a Beckman Acta C-III spectrophotometer. Fluorescence spectra were recorded using an SLM-4800 differential phase fluorometer. Infrared spectra of II were recorded using a Beckman Aculab-2 spectrophotometer.

Mass fragmentography Samples of synthetic nitrobenzdiazole-~0-aminododecanoic acid (II), and concentrated hexane/MeC12 extracts from incubation mixtures, were taken to dryness and submitted for analysis. Mass spectrometric data were obtained using a HP 5985A G C / M S equipped with a data analysis system. The total ion (re~z) mass spectrum (MS) of II was obtained by direct insertion probe (DIP) analysis of the residue using a probe-heating program (50-250°C at 20°C/rain) and an ionizing voltage of 70 eV.

Thin layer chromatography The purity of synthetic II and the identity of products formed during the incubation of I with PLA 2 were checked by thin-layer chromatography using the following solvent systems (all ratios by volume): (1) C H C I 3 / M e O H / H 2 0 (65 : 25 : 4), (2) benzene/pyfidine/glacial acetic acid (80:20:2), and (3) isopropanol/ethylacetate/ammonia (80 : 20 : 4).

Instrumentation for real-time polarization measurements The SLM-4800 fluorometer was modified to facifitate the acquisition of' real-time polarization measurements via the construction of a servo-polarizer linkage which allowed the position of the excitation polarizer to be program-controlled by the HP 9815 calculator. The excitation polarizer rested in a cylindrical housing which could be rotated manually through a 90 ° angle by means of a metal shaft. The shaft was connected to the cylinder housing along an axis perpendicular to the axis of the cylinder. The length traversed by the end of the shaft, when the polarizer rotated

49 SOURCE

i D VER

I EXCITATON ~

II

r I servo-polarlze , ,

EM ISSION POLARIZER

I

V qKRAFT SER 0 ,5,,

EMISSION POLARIZER

Fig. 2. Schematic diagram of SLM-4800 fluorometer and modifications to allow continuous polarization measurements.

through 90 °, was approximately 2 in. Therefore, it was necessary to construct a device capable of moving the shaft through this distance with enough force to rotate the cylindrical housing. These criteria were met by a Kraft 15 II servo obtained from Kraft Radio Corp. An extended arm for the servo was constructed from plexiglas and was coupled to the polarizer shaft by a mechanical linkage. The servo arm would therefore determine the angular position of the excitation polarizer (see Fig. 2). The position of the servo arm could be determined by the width of a pulse, occurring at 20 pulses/s, from the servo driver. A timer-pulse width modulator was constructed to serve as the servo driver in order to allow H P 9815 control of the width of each pulse detected by the servo (see Fig. 3). A N E 556 dual timer (Jameco Electronics) was configured so that one timer, in an astable mode, provided a 20 p u l s e / s trigger to the other timer. Adjustment of the 200-500 k~2 resistor voltage divider (see Fig. 3) regulated the astable timer to produce the necessary 20 Hz trigger. The other timer was configured so that a pulse would be generated with each trigger signal from the astable timer and the width of each pulse would be Vcc =+5

OA

5.6K --

NE556

I

Fig. 3. Schematic diagram of timer-pulse-widthmodulator.

50 determined by the voltage on pins 12 and 13 of the chip (pulse-width modulating monostable). A JE200 5-V power supply (Jameco Electronics) provided the + 5 V needed to operate the timer and servo. The SLM-4800 is capable of monitoring time-dependent intensity changes at fixed wave-lengths. This feature was utilized for processing data and communicating the data to the HP 9815 calculator. The instrument provided internal docking and made data available to the 40 bit input port of the 9815, at specified time intervals. M e a s u r e m e n t o f P L A 2 activity

A 1 ml aliquot of I containing 3.5 nmol was added to 1 ml of 0.2 M phosphate buffer (pH 7.4) in a 3 ml quartz cuvette with a path length of I cm. The temperature was maintained at 23°C by means of a water jacketed sampte holder connected to a temperature programmable Neslab R T E 4 circulating water bath. The contents of the cuvette were stirred continuously via a small magnetic stirring bar in the cuvette and an SLM magnetic stirrer assembly. The excitation wavelength was set at 476 n m and Coming 3-69 filters were inserted in holders between the sample and the two photomultipliers in order to eliminate scattered exciting fight. Slit widths on the excitation monochromator were 4 nm. Following a 5 min interval for temperature equilibration, the channel gain controls were adjusted so that the ratio of the fluorescence monitored by channel A and B was equal to one, with the excitation polarizer parallel to the emission polarizer. Aliquots of PLA 2 in distilled water were rapidly added to the cuvette through an opening in the cover of the sample compartment at t = 0 s, and the data acquisition system was started. In these experiments, a datum point (i.e., the A / B ratio with the excitation polarizer in one of two possible positions) was collected every 5 s. Following the collection of a datum point, the calculator re-oriented the excitation polarizer to the opposite position (e.g., [,, ~ I a , or vice-versa) and another datum point was collected. D a t a collection continued for a total of 1000 s, with the excitation polarizer oscillating through 90 ° between each point. The data points were stored on marked tape files and were recalled for calculating polarization. The calculation of polarization used the two A / B ratios, one with the excitation polarizer perpendicular to channel A's emission polarizer, and the other with the excitation polarizer paralleled to channel A's emission polarizer. The calculated values were then stored on a marked tape file and were recalled for plotting and linear regression analysis. Standard and recovery curves

Standard curves for II were obtained by pipetting aliquots of II into glass tubes and making appropriate dilutions such that, in a final volume of 2.0 ml of MeOH, the solutions contained 0.05-3.0 nmol of II. Recovery curves for II were obtained by pipetfing aliquots of II into glass tubes such that the concentrations of II covered the same range as the standards. Recovery samples were treated (incubation, extraction, etc.) as described above. The overall percent recovery of II was calculated by comparing the fluorescence of the recovery samples, after correcting for dilution, with the fluorescence of samples having the same concentrations of II but which

51 were not put through the entire procedure. Relative fluorescence of II was measured using a Farrand Mark-I spectrofluorometer equipped with a 150 W xenon source. Excitation and emission slit widths were 20 and 10 nm, respectively. The excitation wavelength was 470 n m and emission was monitored at 540 nm. A C o m i n g 3-69 filter was placed between the sample and the emission monochromator to eliminate scattered exciting light.

Determination of pH optlmum The reported p H optimum for PLA 2 from Naja-naja venom is 8.9 [19]. In order to determine whether or not the nitrobenzdiazole moiety of I shifted the p H optimum from that observed with naturally occurring substrate, the hydrolytic activity of PLA 2 on I was measured from p H 6 to 10 in intervals of approximately one p H unit. Stock solutions of 50 n m o l / m l of I were prepared in CHC13. Aliquots of the above solution containing 10 nmol of I were pipetted into glass test tubes (10 × 75 m m ) and were evaporated under N 2. One milliliter of cold 0.2 M phosphate (pH 6-8) or borate (pH 9.3 and 10.0) buffer was added, and the contents of the tubes were vigorously mixed (vortex) for 15 s and sonicated for 5-10 s in a Heat Systems Ultrasonics bath-type sonicator. The last step was necessary to remove the lipid completely from the walls of the tube. Stock solutions containing 20 /~g/ml PLA 2 were prepared fresh for each experiment in distilled water, and were stored at 4°C during the course of each experiment. Blanks, containing no enzyme, were prepared as described above. The reaction was started by addition of enzyme (30 ng) to the sample tubes and all samples and blanks were incubated at 37°C for 30 rain, with gentle shaking. The reaction was terminated by addition of 0.5 ml of 1 N HC1, followed by thorough mixing (vortex) and extraction of the reaction products into 0.5 ml of 1 : 1 ( v / v ) hexane/methylene chloride. The tubes were vigorously mixed (vortexed), centrifuged in a clinical centrifuge, and 100 /zl of the upper phase was applied to T L C plates (Whatman LK6D). The plates were developed using C H C 1 3 / M e O H / H 2 0 (65 : 25 : 4) and were allowed to air dry. Fluorescent bands (two of them) were visualized under U V light and those which were isographic with synthetic II were marked and scraped into glass test tubes. The compounds were eluted from the silica gel with 2.0 ml of methanol. Samples were centrifuged to sediment the silica gel, and aliquots of the supernatants were taken for fluorescence measurements.

Results and Discussion

Characteristics of H The UV-visible spectrum of II showed a m a x i m u m at 465 nm, with an extinction coefficient of 21200 M -1 cm -1. The infrared spectrum of II showed bands at approximately 3460 cm -1 (NH), 1720 cm -1 (C=O), 1590 cm -1 (C=N), and 1290 cm -1 (NO2). Thin layer chromatograms of I and II in three different solvent systems showed a single yellow fluorescent spot for each compound. These data are shown in

52 Table 1. The recovery of 0.05-1.0 nmol of II added to incubation mixtures was linear, with approximately 92% of the added materiat being recovered in a single hexane/methylene chloride extraction. The MS of II showed major mass/charge ( m / z ) peaks at 378 (M+), 361 ( M - 1 7 , loss of - O H ) , 193 ( M - 1 8 5 , loss of -(CH2)10CO2 H) and 161 (M - 217, loss of (CH2)9CO2H and NO2).

p H optimum for PLA 2 actzvzty The hydrolysis of I by phospholipase A 2 as a function of pH was determined as previously described. Optimal enzymatic activity was observed at a pH of approximately 9, in agreement with the reported pH optimum for this enzyme of 8.9 [19]. These results suggest that the presence of the nitrobenzdiazole moiety in I does not significantly shift the optimum pH for PLA 2 activity. In preliminary experiments, it was observed that the values of the blanks incubated at 37°C at p H 9 were approximately 10% higher than corresponding blanks at p H 7. The smallest degree of autohydrolysis of I was observed in blanks incubated at 23°C at pH 7.4 for periods up to 30 rain. Under these conditions, less than 1% autohydrolysis of I occurred. Consequently, all subsequent assays were performed at 23°C and at a p H of 7.4.

Real-time measurement of PLA 2 activity Since the fluorochrome in i is attached to the 2-position of lecithin, hydrolysis of the ester bond at this position would result in the formation of non-fluorescent lysolecithin and a fluorescentty labeled acid. As hydrolysis of I increases, there should be an increase in the formation of II concomitant with a decrease in the amount of I present. This should give rise to two fluorescent species in the cuvette with measurably different rotational rates. The total polarization observed would then depend on both the fractional fluorescence intensity of the two species and their respective rotational mobilities [20]. The data in Eig. 4 show a decrease in observed polarization values following addition of PLA 2 to samples of I. These data are consistent with the interpretation TABLE 1 THIN-LAYERCHROMATOGRAPHYDATA FOR I AND II IN THREE DIFFERENT SOLVENT SYSTEMS Compound I II I II I II

Solvent 1 1 2 2 3 3

Solvent 1:CHCI3/MeOH/H20 (65:25:4). Solvent 2: benzene/pyridine/glacial acetic acid (80 : 20 : 2). Solvent 3: isopropanol/ethyl acetate/NH4OH (80 : 20 : 4).

Rf 0.233 0.723 0.034 0.750 0.081 0.216

53 of time-dependent hydrolysis of I at the 2-position. Under these conditions, additions of PLA 2 containing 14-80 ng protein to incubation mixtures containing 1 ~ M I resulted in a linear decrease in the total measured polarization for approximately 17 rain (1000 s). Additions of PLA 2 containing more than 85 ng protein resulted in a non-linear change in the total measured polarization. The specificity of this response for PLA 2 was determined by adding a 200 ~I aliquot of ovalbumin containing 200/xg protein to a cuvette containing 3/~M [, at t = 175 s. The results, as shown in Fig. 5, indicate that upon the addition of ovalbumin the measured polarization increases by approximately 32%, which would be expected for fluorescent ligand binding to a macromolecule [21]. Additions of the proteolytic enzymes trypsin and pepsin (data not shown) resulted in a similar response. Of the proteins tested, only additions of PLA 2 to samples containing I resulted in a time-dependent decrease in fluorescence polarization. In order to determine whether or not the percent change in polarization is proportional to the percent hydrolysis of I, 10 and 20/~1 aliquots of PLA 2 containing 28 and 56 ng protein were added to samples containing 2 /xM I, at t = 0 s. Total polarization was measured as a function of time for 1000 s, as previously described, and linear least-squares analyses of the data were performed. At the end of 1000 s, the contents of the cuvette were extracted, and the amount of II formed was measured, as previously described. The results shown in Table 2 indicate that under these conditions the observed percent change in polarization is, within experimental error, equal to the percent hydrolysis of I. It is interesting to note that a 2-fold increase in the amount of protein added resulted in only a 1-fold increase in activity. This observation cannot be explained on the basis of substrate depletion, since the plots of total polarization versus time

0.07

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Fig. 4. Representativecurves of total measured polarization (P) as a functzon of time upon addition of 0 (o), 14 (+), and 100 (A) ng of PLA2 to incubation mixtures containing approximately 1/zM I. Enzyme was added at t = 0 s.

54

TABLE 2 COMPARISON OF PERCENT CHANGE IN POLARIZATION WITH DIRECT MEASUREMENT OF HYDROLYSIS PRODUCT AFTER ADDITION OF PLA 2 TO INCUBATION MIXTURES CONTAINING APPROXIMATELY 1 ~M I PLA 2 (ng)

7o z~ P +- S.D.

% Hydrolysis + S.D.

28 56

5.85+_0,83 (N = 3) 7.93 + 0.52 (N = 3)

5.30_+0.92 (N = 3) 7.67 +-0.32 (N = 3)

Enzyme was added at t = 0 s and incubations were carried out for a total of 1000 s. Extractions and calculations were performed as described in the text and values are expressed as the mean_+ standard deviation of three separate determinations.

w e r e l i n e a r o v e r a 1000 s i n t e r v a l a n d e x t r a c t i o n , s e p a r a t i o n , a n d m e a s u r e m e n t o f the contents of the cuvette revealed neither substrate depletion nor abnormally high p r o d u c t f o r m a t i o n . I n fact, as s h o w n i n T a b l e 2, t h e a m o u n t o f p r o d u c t f o r m e d agreed well with the a m o u n t p r e d i c t e d b a s e d on the polarization m e a s u r e m e n t s . T h e s e d a t a m i g h t b e i n t e r p r e t e d as a d d i t i o n a l e v i d e n c e f o r e n z y m e d i m e r f o r m a t i o n , as h a s b e e n s u g g e s t e d f o r P L A 2 [22].

® e

e

e

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o e

e 0.08, e

e e e ~e

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eee

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Fig. 5. Time-dependent increase in polarization upon addition of 200 ~g of ovaibumin to an incubation mixture containing 3/~M I. Ovalbumin was added at t = 175 s. Results are from a single determination.

55

Effects of Ca e + and E G T A

Stimulation of PLA 2 activity by Ca 2+ and inhibition by EGTA has been reported [22]. The data in Fig. 6 indicate that increasing concentrations of Ca 2+ from approximately 2 to 10 /zM result in a greater than 3-fold increase in enzymatic activity. Increasing concentrations of E G T A inhibited Ca 2+ stimulated enzyme activity. In the presence of 2 and 10 ~M EGTA, Ca 2+ (5/xM) stimulation of PLAa activity was reduced by 26 and 57%, respectively. The data in Fig. 9 were used to construct a Hill plot, and calculations based on this plot yielded an apparent Hill coefficient ( n a p p ) of 3.98, suggesting high cooperativity for Ca 2+ interaction with PLA 2. It has been proposed by some investigators that the reactive form of PLA 2 is a dimer [23], and Roberts et al. [24] have suggested a 'dual phospholipid model', where one subunit of the dimer is responsible for activation, while the other subunit catalyzes the hydrolysis of an accessible phospholipid [24]. In the latter model, each mole of enzyme subunit binds one mole of Ca 2+. The data might suggest the possibility that each subunit of the dimer binds two Ca 2 +. However, it should also be pointed out that the enzyme source used in these experiments was not highly purified. Since there are several isozymes of PLA z in Naja-naja venom [25], it would be necessary to repeat the above experiments on purified PLA 2 before any inferences regarding the stoichiometry of Ca 2 + binding to the enzyme can be drawn. The dependence of PLA 2 activity on the physical state of the substrate is well documented [26,27]. However, this method utilizes a fluorescently labeled phospholipid, and additional considerations of the possible effects of the concentration of the fluorochrome on polarization must be made. In the present study, the

41 O

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Fig. 6. Ca 2+ stimulation of PLA 2 activity as measured by polarization assay. Measurements were performed as described in the text. Activity is expressed as the slope of the regression line 0.e. the decrease in polarization/s) obtained from measurement of total polarization as a function of time (1000 s) upon addition of PLA 2 (28 ng) to mixtures containing approximately 1 /zM I, in the presence of increasing Ca 2+ concentrations. Values were corrected for the activity observed in the absence of exogenous Ca 2+, and each point represents the average of two separate determinations.

56 c o n c e n t r a t i o n of lipid was < 3 /xM in o r d e r to avoid possible f o r m a t i o n of lipid aggregates as well as to avoid inner filter effects. This p r o b a b l y m e a n s that the range over which s u b s t r a t e c o n c e n t r a t i o n can b e v a r i e d is fairly narrow. H o w the p h y s i c a t state of the s u b s t r a t e influences P L A 2 activity is an i m p o r t a n t question a n d we are c o n d u c t i n g e x p e r i m e n t s using the m e t h o d d e s c r i b e d here to address this point. The p r e s e n t m e t h o d involves the c o n t i n u o u s coflection of a large n u m b e r of d a t a points. W h i l e it is a d v a n t a g e o u s to b e able to c o n t i n u o u s l y m o n i t o r e n z y m e activity as a f u n c t i o n of time, a certain a m o u n t of precision for each d a t a p o i n t is sacrificed. Therefore, two o r three s e p a r a t e d e t e r m i n a t i o n s (i.e., collections of d a t a ) were m a d e for each c o n d i t i o n tested. D e s p i t e these limitations, the p r e s e n t m e t h o d p e r m i t s the c o n t i n u o u s m e a s u r e m e n t of P L A 2 activity at low e n z y m e a n d s u b s t r a t e concentrations. D a t a can b e g a t h e r e d rapidly, stored, a n d retrieved for further analysis. T h e i n s t r u m e n t m o d i f i c a tions required for this m e t h o d are relatively simple, inexpensive, a n d should b e a d a p t a b l e for m o s t c o m m e r c i a l l y available fluorometers. T h e m e t h o d correctly reflects the k n o w n effects of c o f a c t o r (Ca 2+) a n d a n i n h i b i t o r ( E G T A ) of P L A 2 activity a n d has the p o t e n t i a l for use in kinetic studies of P E A 2. I n a d d i t i o n , the m e t h o d m a y b e e m p l o y e d for s t u d y of the kinetics of l i p i d - p r o t e i n interaction.

Simplified description of the method and its applications Modifications on an SLM 4800 fluorometer are described which allow for automated measurements of fluorescence polarization as a function of time. These modifications included construction of a servopolarizer linkage and a timer-pulse width modulator which allowed the position of the excitation polarizer to be program-controlled by a HP 9815 calculator. Internal clocking and data acquisition features of the fluorometer were utilized for processing data and communication with the calculator, ReaMime fluorescence polarization measurements were used to investigate the interaction of phospholipase A 2 (PLA z) with a fluorescent derivative of phosphatidylcholine (PC). The data indicate that hydrolysis of the labeled PC resulted in a time-dependent decrease m the total measured polarization which was directly proportional to the percent of PC hydrolysis. Calcium stimulated and EGTA inhibited PLAz-induced decrease in polarization. Ca +2 activation appeared to be highly cooperative, and along with other data suggested the possibility that the active form of PLA 2 is a dimer, with each monomer binding two Ca +z. Overall, the results indicate the utility of real-time fluorescence polarization measurements for investigating the interaction of PLA z with suitably labeled fluorescent substrates and suggest the feasibility of applying the technique to the study of other types of lipid-protein interaction.

Acknowledgements T h e a u t h o r s t h a n k Mr. Craig K u e c h e n m e i s t e r for his helpful advice on construction of the servo driver and c o n t r o l units. W e also t h a n k the staff of S L M I n s t r u m e n t s , Inc., for m a n y helpful discussions, a n d Mr. F r a n c o i s B l e a u d e a u for technical assistance. This s t u d y was s u p p o r t e d in p a r t b y a U n i v e r s i t y of A l a b a m a in B i r m i n g h a m F a c u l t y G r a n t (J.A.M.) a n d N I H grant H D l 1 8 9 3 (S.T.C.).

57

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Gilmore, R., Cohn, N. and Glaser, M. (1979) Biochemistry 18, 1042-1049 Goldman, S.S. and Albers, R.W. (1979) J. Neurochem. 32, 1139-1142 Schacter, D., Cogan, U. and Abbott, R.E. (1982) Biochemistry 21, 2146-2150 Cogan, U. and Schackter, D. (1981) Biochemistry 20, 6396-6403 Weber, G. and Bablouztan, B. (1966) J. Biol. Chem. 241, 2558-2561 Spencer, R.D., Toledo, F.B., Williams, B.T. and Yoss, N.L. (1973) Clin. Chem. 19, 838-844 Kelly, R.J., Dandliker, W B. and Williamson, D.E. (1976) Anal. Chem. 48, 846-856 Jolley, M.E., Stroupe, S.D., Wang, C.H.J., (1981) Clin. Chem. 27, 1190-1197 Popelka, S.R., Miller, D.M., Holen, J.T. and Kelso, D.M. (1981) Clin. Chem. 27, 1198-1201 Monti, J.A., Christian, S.T., Shaw, W.A. and Finley, W.H. (1977) Life Sci. 21, 345-356 Monti, J.A., Sarnf, A.M., Christian, S.T. and Saxholm, M.J.K. (1979) Arch. Biochem. Biophys. i93, 496-501 Monti, J.A., Christian, S.T. and Shaw, W.A. (1978) J. Lipid Res. 19, 222-228 Wells, M.A. (1972) Biochemistry 11, 1030-1036 Shakir, K.M.M. (1981) Anal. Biochem. 114, 64-70 Carman, G.M., Fischl, A.S., Dougherty, M. and Maerker, G. (1981) Anal. Biochem. 110, 73-76 Hendrickson, H.S. and Rauk, P.N. (1981) Anal. Biochem. 116, 553-558 Wolf, C., Sagaret, L. and Berezmt, G. (1981) Biochem. Biophys. Res. Commun. 99, 275 283 Fager, R.S., Kutina, C.B. and Abrahamson, E.W. (1973) Anal. Biochem. 53, 290-294 McMurray, W.C. and Magee, W.L. (1972) Annu. Rev. Biochem. 41, 129-160 Weber, G. (1952) Biochem. J. 51, 145-155 Anderson, S.R. and Weber, G. (1969) Biochemistry 8, 371-377 Long, C. and Penny, I.F. (1957) Biochem. J. 65, 382-389 Deems, R.A. and Dennis, E.A. (1975) J. Biol. Chem. 250, 9008-9012 Roberts, M.F.. Deems, R.A. and Dennis, E.A. (1977) Proc. Natl. Acad. Sci. U S.A. 74, 1950-1954 Brocherhoff, H. and Jensen, R.G. (1974) in Lipolytlc Enzymes, Ch. 6, p. 208, Academic Press, New York Menashe, M., Lichtenberg, D., Guuerrez-Merino, C. and Biltonen, R.L. (1981) J. Biol. Chem. 256, 4541-4543 Dennis, E.A., Darke, P.L., Deems, R.A., Kensil, C.R. and Pluckthun, A. (1981) Mol. Cell. Biochem. 36, 37-45