Analysis of Activation-induced Conformational Changes in p47 phox Using Tryptophan Fluorescence Spectroscopy*

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47 phox , p67 phox , and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47 phox undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47 phox using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47 phox with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47 phox , whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47 phox fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47 phox . Protein kinase C phosphorylation of p47 phox in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47 phox was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47 phox . Thus, our studies provide direct evidence linking conformational changes in p47 phox to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47 phox that are similar to those mediated by phosphorylation in vivo.

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47 phox , p67 phox , and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47 phox undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47 phox using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47 phox with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47 phox , whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47 phox fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47 phox . Protein kinase C phosphorylation of p47 phox in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47 phox was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47 phox . Thus, our studies provide direct evidence linking conformational changes in p47 phox to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47 phox that are similar to those mediated by phosphorylation in vivo.
Human neutrophils play an essential role in host defense and are key participants in the inflammatory response to foreign pathogens (for reviews, see Refs. 1 and 2). During the inflammatory response, neutrophils are readily mobilized to sites of inflammation, where they phagocytose pathogens and/or fragments of damaged tissue and subsequently release a variety of toxic oxygen radical species which can destroy the engulfed material (1,2). The generation of oxygen radicals by neutrophils is known as the respiratory burst and results from the activation of a membrane-bound NADPH oxidase (reviewed in Refs. [3][4][5]. During the respiratory burst, electrons are transferred from NADPH to molecular oxygen, producing superoxide anion (O 2 . ), the direct product of the NADPH oxidase. O 2 . is then rapidly converted to secondary toxic oxygen species such as hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH ⅐ ), and hypochlorous acid (HOCl) that can efficiently kill microorganisms; in combination with the primary and secondary granule contents, O 2 . constitutes the primary host defense mechanism used by neutrophils (6). The NADPH oxidase is a highly regulated enzyme complex that is composed of a number of cytosolic and membrane-bound proteins (reviewed in Refs. [3][4][5]. In the resting cell, these proteins remain segregated into cytosolic and plasma membrane compartments. However, because the active enzyme complex is located on the plasma membrane, the cytosolic proteins must translocate from the cytosol to the membrane during assembly of the functional enzyme complex (3)(4)(5)7). This translocation process is initiated by a series of highly regulated signaling events, resulting in assembly of the active complex.
The mechanism by which the NADPH oxidase cytosolic proteins assemble during activation of the oxidase is complex and not completely understood. One of the key cytosolic proteins in this process is known as p47 phox , which seems to be the first cytosolic component to interact with flavocytochrome b during the assembly process (7)(8)(9), and its association with flavocytochrome b is a prerequisite for binding of p67 phox and/or p40 phox (8 -10). During NADPH oxidase activation, p47 phox has been shown to undergo extensive phosphorylation (11)(12)(13)(14)(15), and agents that stimulate phosphorylation of p47 phox in intact neutrophils (such as phorbol esters) can also stimulate O 2 . production (16). Furthermore, inhibition of phosphorylation during neutrophil activation by the protein kinase C (PKC) 1 inhibitor staurosporine markedly decreases both O 2 . generation and translocation of p47 phox and other cytosolic oxidase proteins in vivo (12,17). Subsequent studies of the exact sites of phosphorylation in p47 phox confirmed that p47 phox serines 303, 304, 320, 328, 345, 348, and 379 were phosphorylated during activation of the oxidase (14,15,18,19). These observations support the conclusion that phosphorylation of p47 phox is a necessary event in the activation of the NADPH oxidase in vivo.
In contrast to intact neutrophils, production of O 2 . in the cell-free assay system does not require phosphorylation of p47 phox (20,21), although there is a requirement for anionic amphiphilic detergents such as sodium dodecyl sulfate (SDS) or arachidonic acid (22)(23)(24). It is still not clear whether amphiphile activation in vitro is mimicking actual physiological events in vivo, but it has been suggested that activation with amphiphiles may share mechanistic similarities with activation by phosphorylation (5,23). In support of this idea, it has recently been shown that PKC can also induce O 2 . generation in the cell-free system without added amphiphiles, thus mimicking the phosphorylation-dependent activation process occurring in vivo (25,26). It has been proposed by several investigators that phosphorylation causes a conformational change in p47 phox and/or neutralizes the cationic region of the protein in vivo so that it may interact with the membrane (15,20). Thus, in a similar manner, SDS or arachidonic acid may provide a neutralizing negative charge which allows p47 phox to undergo similar conformational changes in vitro that are likely to occur with phosphorylation in vivo (22)(23)(24)27). p47 phox contains a total of seven tryptophan residues. Interestingly, five of the tryptophans are located in the region of p47 phox containing both Src homology 3 (SH3) domains (28,29) and the cationic flavocytochrome b/p67 phox binding domain (30,31), all of which appear to be masked in the resting cell. In addition, Sumimoto et al. (32) recently reported that mutation of a tryptophan (residue 193) to arginine in the first SH3 domain of p47 phox resulted in a nonfunctional p47 phox , possibly by altering the charge distribution in this key functional domain of p47 phox . Thus, we hypothesized that if activation of the NADPH oxidase induced unmasking of this region of p47 phox (28 -31), then significant and measurable changes would occur in the micro-environment of these tryptophan residues.
In the present studies, we measured changes in p47 phox tryptophan fluorescence before and after exposure to several in vitro activators of NADPH oxidase. We present data showing that both amphiphiles (SDS and arachidonic acid) and PKC phosphorylation cause similar changes in the inherent tryptophan fluorescence of p47 phox , and these changes in fluorescence correlated directly with NADPH oxidase activity. Thus, our results provide direct evidence linking conformational changes in p47 phox to the activation process of the neutrophil NADPH oxidase.

MATERIALS AND METHODS
Preparation and Fractionation of Neutrophils-Purified human neutrophils, isolated as described previously (33), were disrupted by nitrogen cavitation, and membrane and cytosolic fractions were prepared from this cavitate using sequential centrifugation, as described by Fujita et al. (34).
Purification of Recombinant p47 phox -Recombinant p47 phox was produced in baculovirus-infected Sf9 cells using a modification of the method of Leto et al. (35). Briefly, Sf9 cells were infected with recombinant baculovirus at a multiplicity of infection of 10. After 72 h, the cells were collected, disrupted by cavitation, and the cytosol collected by differential centrifugation (35). Diluted supernatant from ϳ2-4 ϫ 10 8 cells was applied to a Bio-Rad Macro-Prep CM column (10 ml bed volume), the column was washed, and p47 phox was eluted by application of a 0 -0.3 M NaCl gradient in 5 mM potassium phosphate, pH 7.0. The purity (Ͼ95%) and identity of the recombinant p47 phox were confirmed using SDS-polyacrylamide gel electrophoresis and Western blotting with an anti-p47 phox monoclonal antibody (31). Protein content of the fractions was determined using a Pierce BCA assay.
Fluorescence Assays-For fluorescence analysis, recombinant p47 phox (in a volume of 40 -70 l) was added as indicated to a final volume of 2 ml of cell-free assay buffer (10 mM potassium phosphate, pH 6.7, 130 mM NaCl, 1 mM EGTA) in a quartz cuvette containing a stir bar. The samples were then analyzed on a Photon Technology International scanning fluorimeter at 25°C using an excitation wavelength of 280 nm (1-nm bandwidth) and recording the emission spectra from 300 to 450 nm (2-nm bandwidth). Although measurement of tryptophan fluores-cence is often performed using an excitation wavelength of 295 nm instead of 280 nm to reduce the contribution of tyrosine residues to the signal, we found that absolute fluorescence of tryptophan was significantly reduced at 295 nm, and the signal to noise ratio using small amounts of protein was much lower. Thus, to confirm that we were only measuring tryptophan fluorescence, we performed a dose-response of fluorescence versus SDS concentration using excitation at both 280 and 295 nm and found that the dose-response curves plotted as a percentage of control peak fluorescence were identical for the two different excitation wavelengths (data not shown), indicating that tyrosine fluorescence did not contribute significantly to the observed signal.
Excitation at 280 nm with 2-5-nm bandwidths led to a slight photobleaching of p47 phox (approximately 5%); therefore, the excitation bandwidth was kept at a minimum to eliminate photobleaching artifacts. Once a base-line spectrum of the protein sample was obtained, SDS or arachidonic acid (or other agents as described under "Results") could be added directly to the cuvette (with stirring) and a new spectrum obtained immediately. SDS and arachidonic acid were added from concentrated stocks to the final concentrations indicated under "Results." The arachidonic acid stock solution contained 25% ethanol, but controls containing the ethanol alone did not change the fluorescence spectrum of the protein (maximum final ethanol concentration was 0.4%) (data not shown).
In Vitro Phosphorylation of Recombinant p47 phox -Nine g of purified p47 phox were added to a buffer containing 10 mM PIPES, pH 7.3, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl 2 , 0.5 mM CaCl 2 , and 1 mM ATP. Three sets of samples, each in triplicate, were used to compare the fluorescence of p47 phox phosphorylated by PKC (with activators), p47 phox with inactive PKC (without activators), and the fluorescence of the PKC and activators alone. The first set contained 0.2 unit of PKC, 25 g of phosphatidylserine, and 3 g of dioctanoyl glycerol. The second set contained 0.2 unit of PKC, but no phosphatidylserine or dioctanoyl glycerol. The third set contained 0.2 unit of PKC, 25 g of phosphatidylserine, and 3 g of dioctanoyl glycerol, but no p47 phox . Phosphatidylserine and dioctanoyl glycerol were added in the form of mixed micelles produced as follows. Solutions of the lipids in chloroform were added to a small glass tube and dried down to a lipid film under nitrogen at 4°C. The lipids were resuspended in 20 mM Tris, pH 7.4, by vortexing, sonicating for 2 ϫ 3 s, transferring to a polypropylene microcentrifuge tube, and sonicating again for 10 ϫ 3 s. Twenty l of this suspension was added to the relevant samples, and the final reaction volume for all samples was 80 l. Samples were placed in a 37°C bath for 20 min. After incubation, the entire volume of the reaction was added to 1.92 ml of cell-free assay buffer and analyzed fluorometrically as described above.
To verify that phosphorylation was taking place under these conditions, the above ingredients were mixed in the same proportions, but in a smaller final volume (30 l), and 1 mM unlabeled ATP supplemented with 3 Ci of [␥-32 P]ATP was added per tube. After incubation at 37°C for 20 min, the samples were washed of free ATP by filtering the sample through a Spinzyme phosphocellulose filter (Pierce), and the filter with bound protein was placed into a scintillation vial and counted using a Packard scintillation counter. Three sets of duplicate samples were analyzed: one with the complete mixture, one without the lipid activators, and one without p47 phox .
Analysis of p47 phox Phosphorylation by Two-dimensional Gel Electrophoresis and Western Blotting-Normally, p47 phox has been analyzed in the past using non-equilibrium pH gradient electrophoresis (NEPHGE) because of its highly basic nature (36). However, studies by Görg et al. (37) have shown that basic proteins normally separated by NEPHGE with limited reproducibility can now be perfectly separated under equilibrium conditions using immobilized pH gradients for isoelectric focusing. Therefore, we utilized this method for analyzing p47 phox .
Phosphorylation reactions were performed as described above; however, the reactions were stopped by the addition of 225 l of Immobiline rehydration buffer (8.0 M urea, 2% CHAPS, 0.5% Pharmalyte 3-10, and 0.3% DTT). The samples were then placed in individual slots of an Immobiline reswelling tray with an Immobiline 3-10L DryStrip (Pharmacia Biotech, Uppsala, Sweden). The strips were allowed to swell overnight and were then subjected to isoelectric focusing in a Multiphor II electrophoresis apparatus. Isoelectric focusing was done stepwise (1 h at 300 V, 13 h at 1000 V, at 3.5 h at 2000 V) for a total of 20,300 V⅐h. The focused strips were frozen until the second dimension was run. To run the second dimension, the strips were thawed, gently agitated in equilibration buffer with DTT (100 mM Tris, pH 6.8, 6 M urea, 1% SDS, 30% glycerol (v/v), and 0.2% DTT) for 2.5 min and then in equilibration buffer without DTT for 2.5 min. The strips were then placed on a 10% SDS-polyacrylamide gel and overlaid with a small amount of 0.5% agarose in stacking gel buffer. The gel was then run, followed by transfer of the proteins to nitrocellulose by electroblotting. Phosphorylated and non-phosphorylated p47 phox spots were detected by probing with monoclonal anti-p47 phox , as described previously (31). Because the Immobiline strips have an immobilized linear pH gradient, we could estimate the pI of the p47 phox spots from their relative horizontal position on the blot. Relative spot densities were quantified by densitometric analysis with an IS-1000 digital imaging system (Alpha Innotech, San Leandro, CA).
Cell-free Assay of NADPH Oxidase Activity-Amphiphile-activated NADPH oxidase activity was measured spectrophotometrically in a cell-free NADPH oxidase assay system as described previously (38). Briefly, 200 l assays containing 10 mM potassium phosphate, pH 6.7, 130 mM NaCl, 1 mM EGTA,, 10 M FAD, 2 mM NaN 3 , 50 M cytochrome c, 4 l of neutrophil membranes, 4 l of neutrophil cytosol, and 10 M GTP␥S were incubated with SDS or arachidonic acid (at concentrations as indicated under "Results") for 5 min, followed by the addition of 200 M NADPH to initiate the reaction. The change in absorbance at 550 nM was measured continuously over 15 min at 25°C in a Molecular Devices THERMOmax microtiter plate reader. Results are presented as V max rates, calculated using an ⑀ ϭ 1.88 mM Ϫ1 cm Ϫ1 for cytochrome c, and expressed as nanomoles of O 2 . /min/mg of membrane protein.
A variation of the method of El Benna et al. (26) was used to measure O 2 . production in a PKC-activated cell-free assay. We used a microtiter plate assay with 0.2-ml reaction volumes containing 10 mM PIPES, pH 7.3, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl 2 , 0.5 mM CaCl 2 , 0.4 mM ATP, 50 M GTP␥S, 4 l of neutrophil cytosol, and 10 l of neutrophil membranes. In some experiments, recombinant cytosolic proteins were substituted for cytosol as follows: 3-5 g of p47 phox , 3-5 g of p67 phox , and 0.5 g of Rac2 (production of the latter two proteins is described in Ref. 31). Lipid activators of PKC were added in the form of mixed micelles of phosphatidylserine (25 g/well) and dioctanoyl glycerol (3.3 g/well), and micelles were made as described above. PKC (0.2 units) was added to each well, the plates were incubated for 5 min at 37°C, and the reactions were initiated by the addition of cytochrome c and NADPH at final concentrations of 0.1 and 0.2 mM, respectively. O 2 . production was measured as described above, except that the plate reader was maintained at 37°C. Circular Dichroism (CD) Spectroscopy-For CD spectroscopy, samples of p47 phox were dialyzed into a low ionic strength buffer (15 mM NaCl, 10 mM sodium phosphate, pH 7.1) to minimize the UV absorbance of the buffer below 195 nm. SDS or Zwittergent 3-14 were added to the samples and buffer controls as indicated at final concentrations of 150 M. The far UV circular dichroism spectra of the p47 phox samples were measured in 0.02-cm pathlength cuvettes using a JASCO model J-710 CD spectrometer. CD spectra were recorded from 260 to 185 nm at a digital resolution of 0.5 nm, with scan speeds of 5 and 2 nm/min for wavelengths above and below 194 nm, respectively. Eight scans were signal averaged for each wavelength range, and the resulting data sets were concatenated. The final CD spectrum was obtained by subtracting base-line scans of the buffer blank measured in the same cuvette used for the sample. To correct the measured CD intensities for the absorption flattening effects (39) observed in protein samples containing SDS (see "Results"), the far UV absorption spectra from 260 -185 nm were measured in the same instrument, and the absorption flattening coefficients were calculated from the ratio of the UV absorption spectra measured with and without detergent present.

Effects of Amphiphile Treatment on p47 phox Fluorescence and
NADPH Oxidase Activity-To analyze potential activation-induced conformational changes in p47 phox , we measured its intrinsic tryptophan fluorescence before and after exposure to agents that are used as activators of the NADPH oxidase in the cell-free assay system. Serial additions of concentrated stocks of SDS were made to a stirred sample of p47 phox , and the emission spectrum was scanned after each addition. A typical set of fluorescence spectra obtained by mixing various amounts of SDS with purified recombinant p47 phox is shown in Fig. 1  (left panel). Over the range of 50 -200 M SDS, each addition of SDS resulted in a decrease in the tryptophan fluorescence intensity, with a maximum decrease of 67% at 200 M SDS. The emission peak for the tryptophan fluorescence at 335 nm was not shifted by the addition of SDS. This decrease in p47 phox fluorescence observed after the addition of SDS was also very stable. If scans are repeated every minute for 40 min after the addition of SDS, the peak fluorescence intensity did not increase at all back toward the pretreatment level. In fact, there was actually a slight (ϳ5%) but gradual drop in fluorescence over this time, possibly as a result of further photobleaching because of the large number of scans (data not shown).
Addition of another commonly used cell-free oxidase activating agent, arachidonic acid, to p47 phox also resulted in a decrease in the p47 phox tryptophan fluorescence spectrum, which was very similar to that induced by SDS; the fluorescence peak of p47 phox dropped over the range of 5-50 M arachidonic acid and remained at a similar level over the range of 50 -125 M arachidonic acid (see Fig. 1, center panel). Interestingly, at higher concentrations of arachidonic acid, we observed a small amount of background fluorescence from arachidonic acid alone (peak at ϳ320 nm). This background fluorescence was probably a result of some impurities in the arachidonic acid, and the same fluorescence pattern was observed with arachidonic acid from several sources. In any case, we eliminated the contribution of this background fluorescence in our studies by subtracting the spectra from the corresponding concentrations of arachidonic acid alone from that of p47 phox plus arachidonic acid to determine the effect of arachidonic acid on p47 phox fluorescence.
Several other detergents were also tested for their ability to alter p47 phox tryptophan fluorescence, including CHAPS (0.01-  Fig. 1, right panel). Note that all of these detergents failed to activate the cell-free NADPH oxidase system in our present studies and as reported previously for CTAB, Tween 20, and sodium deoxycholate (23). Consistent with this lack of activating capacity, no change in the p47 phox fluorescence (with the exception of a slight (Ͻ5%) decrease as a result of sample dilution) was seen after the addition of CHAPS, Tween 20, sodium deoxycholate, or Zwittergent 3-14.
Interestingly, CTAB at concentrations Ͼ100 M caused a redshift in the peak fluorescence from 335 nm to 341 nm and a modest (ϳ8 -15%) reduction in peak fluorescence intensity, suggesting that higher concentrations of CTAB caused nonspecific denaturation of the protein. Consistent with this result, the addition of 8 M urea to denature the protein also caused a red-shift (ϳ5 nm) in the p47 phox emission peak compared with that observed for intact p47 phox without quenching peak fluorescence intensity (data not shown).
Activation of the NADPH oxidase by SDS in a cell-free assay system resulted in a biphasic dose-response curve for O 2 . production, as has been reported previously (23,40,41). In our study, maximal O 2 . production occurred at an SDS concentration of 125 M (see Fig. 2A). There are other reports of maximal O 2 . production occurring at lower SDS concentrations (23); however, in most of these studies, the membrane preparations contained some amount of another detergent (e.g. sodium deoxycholate), which may have had synergistic effects with SDS. Investigators that did not appear to use detergents in the preparation of their membrane suspensions had dose-response curves similar to ours (40). As shown in Fig. 2B, activation of the NADPH oxidase by arachidonic acid in the cell-free assay also showed a similar dose-response curve; however, arachidonic acid is an effective stimulator at a lower concentration than SDS, it has a steeper threshold of activation, and it induced a much higher level of O 2 . production. As with SDS activation, there is some discrepancy in the literature regarding the optimal arachidonic acid concentrations for NADPH oxidase activation (22,40). This is probably a result of differences in preparations of neutrophil membranes, as discussed above, or to different assay conditions. What is strikingly apparent in Fig. 2 is that there is an inverse relationship between the change in fluorescence of p47 phox and the change in NADPH oxidase activity over a particular range of activator concentrations. When SDS is used as an activator, O 2 . production increases over a range of SDS concentrations from 30 -125 M. Over the same range of SDS concentrations, p47 phox tryptophan fluorescence is decreasing. With arachidonic acid activation, O 2 . production rises rapidly as arachidonic acid concentration is increased from 0 -25 M, whereas in the same concentration range p47 phox fluorescence is decreasing. Thus, even though SDS and arachidonic acid stimulate O 2 . production in different concentration ranges, the increase in oxidase activity directly coincides with the decrease in p47 phox tryptophan fluorescence, suggesting that p47 phox undergoes conformational changes during activation of the NADPH oxidase. At higher amphiphile concentrations (Ͼ125 M SDS or Ͼ25 M arachidonic acid), the decrease in tryptophan fluorescence plateaus, yet a decrease in NADPH oxidase activity is observed. This could be caused by possible denaturation of one or more of the NADPH oxidase proteins. For example, flavocytochrome b and p67 phox are both highly sensitive to denaturation (42,43). A second possibility is that there is an optimal range in p47 phox conformational change (or neg-ative charge accumulation as a result of amphiphile binding) that facilitates oxidase activation and that further changes can result in decreased activity. In support of this idea, Yamaguchi et al. (44) found that hyperphosphorylated p47 phox lost its ability to activate the NADPH oxidase. Because the amount of protein present in the cell-free assays was ϳ10-fold higher than that used in the fluorescence measurements, thereby making the detergent to protein ratios different, we repeated the fluorescence assays with higher amounts of protein. As shown in Fig. 3, the shape of the doseresponse curves was the same if either 9 or 90 g of protein was present in the cuvette, although the magnitude of the shift was slightly less when the higher amount of protein was used. Thus, the effects on p47 phox fluorescence seem to be more a function of the absolute detergent concentration, and not the ratio of protein to SDS. As shown in Figs. 1 and 2, the decrease in p47 phox fluorescence occurred over a range of 50 -200 M SDS. These concentrations are far below the critical micelle concentration (CMC) of SDS, which we measured in our buffer system to be 1-1.5 mM using a fluorometric method (45). Thus, SDS is used at concentrations that are nowhere near the range where phase transitions would occur. In contrast, the CMC of arachidonic acid has been reported to be 10 M (27), which is in the range where we see the major changes in p47 phox fluores- cence. However, these authors also found that CMC is not a factor involved in activation of the cell-free assay by arachidonic acid (27). Additionally, to compensate for any solution effects as a result of arachidonic acid phase transition in the fluorescence assay, we have subtracted out background signals arising from arachidonic acid alone (see above). Finally, we also saw no solution effects using a number of inactive detergents at concentrations spanning their CMCs (see above). Taken together, these results indicate that the changes in p47 phox fluorescence reported here are caused by effects on the protein itself and are not simply the result of detergent phase transitions.
Effects of Phosphorylation on p47 phox Fluorescence and NADPH Oxidase Activity-Although activation of the cell-free NADPH oxidase assay system with amphiphiles results in O 2 .
production, there are various aspects of this cell-free assay that differ significantly from physiological activation of the NADPH oxidase in vivo. For example, various steps required for in vivo NADPH oxidase activation, such as p47 phox phosphorylation, are not essential in the cell-free assay system (15,20,46,47). Therefore, we analyzed NADPH oxidase activity and p47 phox tryptophan fluorescence using a PKC-activated cell-free system, which provides a closer representation of NADPH oxidase activation in vivo. In this case, PKC-mediated phosphorylation, rather than the addition of SDS or arachidonic acid, was used as the activating event. As shown in Table I, PKC-mediated phosphorylation resulted in rates of O 2 . production that were significantly higher than controls. This is similar to what has been shown by El Benna et. al. (26). This assay requires greater amounts of purified neutrophil membranes for O 2 . generation, but cytosolic components (including p47 phox ) are included in the same proportions as in the amphiphile-activated assays. Because phosphorylation in vitro is an all or none phenomenon, we could not obtain a dose-response curve of phosphorylationinduced changes in p47 phox fluorescence. Instead, samples of p47 phox were mixed with PKC and the appropriate activators, incubated for 20 min at 37°C, and the entire sample was then added to a cuvette for fluorescent analysis. A duplicate set of samples without the lipid activators (hence without phosphorylation) was treated the same way, as were blanks containing PKC and activators alone. The fluorescence spectrum of the blanks was subtracted from that of phosphorylated and nonphosphorylated p47 phox , and the composite spectra (each curve representing three replicates with Յ2% error between the replicates) are shown in Fig. 4. Phosphorylation of p47 phox resulted in approximately a 42% reduction of tryptophan fluorescence. This level of quenching is similar to that which was caused by SDS or arachidonic acid when they were present at concentrations that supported near-maximal rates of O 2 . production in the cell-free assay system. To confirm that p47 phox was phosphorylated, parallel experiments were performed using [ 32 P]ATP in the reaction mixture. Under these conditions, p47 phox was phosphorylated with a stoichiometry of 6.8 Ϯ 0.1 mol of P i /mol of p47 phox (mean Ϯ S.E.; n ϭ 4), whereas less than 10% (0.7 Ϯ 0.2 mol of P i /mol of p47 phox (mean Ϯ S.E.; n ϭ 4)) of this level of phosphorylation was observed if the lipid activators (phosphatidyl serine and diacylglycerol) were omitted from the reaction. This level of p47 phox phosphorylation is consistent with that reported previously by El Benna et al. (26) using similar conditions. In addition, we performed two-dimensional gel electrophoresis followed by Western blotting for p47 phox to confirm phosphorylation and evaluate for the presence of the acidic, phosphorylated species of p47 phox , which have been well described for p47 phox (36). In phosphorylated samples, Ͼ90% of the staining was represented by three distinct spots with pI values of ϳ6.5, 6.6, and 6.8 (blots not shown). This is the region shown previously (36) to contain the most acidic and, therefore, the most highly phosphorylated forms of p47 phox . We also found a small spot at a pI of ϳ4.7 (9% of the total staining), which may represent a highly phosphorylated form of p47 phox that was not identified previously because of the low resolution of standard isoelectric focusing gels in this pI range. Consistent with the studies described above using 32 P, no non-phosphorylated p47 phox (pI ϳ8.6) was observed in these samples. In contrast, the majority of staining in control samples treated with PKC alone (no lipid activators) was present as a single spot at a pI of ϳ8.6 (85% of the staining on the blot) with a small amount of phosphorylated species represented by a single spot at a pI of ϳ6.7 (ϳ15% of the staining), which most likely represents a low level of PKC activity even in the absence of lipids. This is also consistent with our results showing a low level of 32 P labeling in control samples. In any case, these results confirm that the fluorescence changes observed in p47 phox did indeed result from PKC-mediated phosphorylation.
One of the lipid activators of PKC used in this assay (dioctanoyl glycerol) has been shown to have some potential for activating O 2 . production in a cell-free assay system (48). As  shown in Table I, control reactions containing the lipid activators without PKC were slightly higher than controls with SOD but were not significantly different from control reactions containing PKC with no lipids (Table I). We also tested the effect of dioctanoyl glycerol on tryptophan fluorescence of p47 phox and found that, at concentrations equivalent to those used in the cell-free assay, there was only a modest (ϳ15%) decrease in fluorescence at 335 nm (data not shown). Thus, the results suggest that if dioctanoyl glycerol can cause a similar effect on protein conformation as SDS or arachidonic acid, it is of a much smaller magnitude when used at the concentrations reported here.
Effects of Amphiphile Treatment on the CD Spectrum of p47 phox -To further analyze amphiphile-induced conformational changes in p47 phox , we measured the CD spectrum of p47 phox before and after treatment with SDS. We used a Zwittergent 3-14 as a control detergent because it has a similar structure to SDS and because it had no effect on the p47 phox fluorescence spectrum. As shown in Fig. 5A, the far UV CD spectrum of p47 phox exhibits a positive maximum of 188 nm and a broad minimum at 207 nm. The zero crossing between these bands was near 195 nm. The far UV absorption spectrum of this sample had a maximum at 190 nm with an optical density of 0.48 measured in the 0.02 cm pathlength cell (data not shown). Neither the CD spectrum nor the UV absorption of the protein was changed by the addition of 150 M Zwittergent 3-14 (Fig. 5A). In contrast, the CD spectrum of p47 phox was dramatically affected by the addition of 150 M SDS (Fig. 5B). The intensities of both the positive and negative bands decreased, and the zero crossing was red-shifted. However, the far UV absorption of the protein was also lower in the presence of the detergent, with the optical density decreasing by approximately 30% at 190 nm (data not shown).
To compensate for absorption flattening effects that can arise from detergent-induced aggregation of proteins (see "Discussion"), the absorption flattening coefficients calculated from the flattened versus control absorption spectra were used to correct the distorted CD spectrum (see "Materials and Methods"). The corrected CD spectrum shows that the intensity of the positive maximum at 188 nm was actually not decreased by the addition of SDS; however, the negative maximum was still decreased by approximately 10% (Fig. 5B). In addition, many other differences between the spectra observed for the SDStreated and untreated samples (e.g. shift in zero crossing, etc.) were maintained even after correction for flattening effects, supporting the validity of our CD measurements. Note that the same correction procedure applied to the CD spectrum of p47 phox measured in the presence of Zwittergent 3-14 did not change the spectrum at all (Fig. 5A). This would be expected because the UV absorption spectrum of p47 phox was not affected by this detergent. DISCUSSION Although the requirement for p47 phox in the neutrophil NADPH oxidase is well documented (reviewed in Ref. 49), the precise role played by p47 phox in the oxidase activation and assembly process is still uncertain. The molecular interactions occurring between p47 phox and the other NADPH oxidase proteins are spatially and temporally complex; however, some details of the molecular basis of interaction of the oxidase components are becoming clear. During activation of the neutrophil NADPH oxidase, p47 phox binds directly to flavocyto- chrome b at multiple peptide domains on both subunits (8,9,50), and at least six regions of flavocytochrome b have been identified as binding sites for p47 phox (9,28,29,(51)(52)(53). In addition, p47 phox is known to bind directly to other NADPH oxidase proteins, including p67 phox and p40 phox . Finally, p47 phox and p67 phox each contain two SH3 regions, which have been shown to mediate the binding interactions between p47 phox and p67 phox and between p47 phox and the proline-rich carboxyl-tail of p22 phox (29).
In vivo, the interactions of p47 phox with these other oxidase components seems to be dependent on phosphorylation. There are multiple potential phosphorylation sites in the carboxylterminal region of p47 phox , which is believed to be an important region for interaction with other oxidase proteins (14,15). p47 phox has been shown to exist in multiple phosphorylation states in vivo, and there seems to be a relationship between the phosphorylation state of p47 phox and the assembly and activation of the oxidase (11). In resting neutrophils, p47 phox is not phosphorylated (11). During the first 30 s after activation, only the four most acidic forms of p47 phox are associated with the membrane fraction, whereas all the forms of p47 phox can be found associated with the membrane fraction after 5-15 min (11). These results suggest that, during activation in vivo, there is a specific sequence of phosphorylation events and subsequent interactions with the membrane and cytosolic components that results in NADPH oxidase assembly. In contrast to the situation in vivo, phosphorylation of p47 phox is not required for activation of the oxidase in vitro. In the cell-free assay system, neutrophil components are commonly activated to produce O 2 . by the addition of an amphiphile, such as SDS or arachidonic acid (40). Whether this mode of activation has anything in common with activation by phosphorylation is still in question; however, it has been proposed that if phosphorylation is required to neutralize the highly cationic carboxylterminal region of p47 phox and allow it to interact with potential binding sites on either p67 phox or p22 phox , then the association of an anionic amphiphile such as SDS or arachidonate may also serve the same purpose by imparting negative charge to this region of the protein.
Based on a number of recent studies, it seems likely that p47 phox undergoes conformational changes during the process of NADPH oxidase activation to facilitate the series of interactions with the other oxidase components. Sumimoto et al. (28) found that the sites in p47 phox that bind to p67 phox and p22 phox are masked within the p47 phox molecule before activation and that some sort of conformational change must be induced in p47 phox by the addition of arachidonic acid, resulting in the unmasking of these sites; recent studies by de Mendez et al. (54,55) using deletion analysis of oxidase proteins in transfected K562 cells support this conclusion. Finally, we recently identified an activation-dependent interaction between p67 phox with p47 phox and showed that p67 phox binds to a functional domain of p47 phox , which is later occupied by a flavocytochrome b (31). This mutually exclusive binding site is located within the cationic domain of p47 phox and encompasses residues 323-332. Thus, it is clear that conformational changes occur in p47 phox during the activation process; however, the exact nature of these changes is still a mystery. Detailed conformational analysis of proteins is of course a difficult and expensive process. One method that is relatively easy, although not definitive in nature, is analysis of the intrinsic tryptophan fluorescence of a protein. Tryptophan residues stimulated at a wavelength of 280 will emit fluorescence in the 300 -400 nm range. The magnitude of this fluorescence in a protein is normally a function of the number of tryptophan residues and their immediate environment in the protein folds (i.e. whether the residues are relatively exposed or buried) (56). The specific wavelength of the peak may also shift as the tryptophan residues change to a more or less polar environment in the protein folds (57,58). In the present studies, we utilize tryptophan fluorescence to investigate putative conformational changes in p47 phox and also use this method to analyze whether a similar conformational change would be induced in p47 phox whether it was activated by PKC phosphorylation or by addition of amphiphiles. Our results show that conformational changes (as demonstrated by a quenching of the tryptophan fluorescence) occurred in p47 phox treated with SDS and arachidonic acid, known activators of the oxidase in vitro, but not in p47 phox treated with several other amphiphilic molecules that do not activate the oxidase. Furthermore, the concentration range of amphiphilic agents required to induce changes in fluorescence correlated directly with the concentration range of these activating agents that induced maximal NADPH oxidase activity in a cell-free assay system. These results suggest that the change in activity of the oxidase, which depends on the association of the oxidase components, may in turn be dependent on a specific conformational change in p47 phox indicated by the quenching of tryptophan fluorescence. We then asked the question of whether activation by phosphorylation causes the same type of conformational change in p47 phox . Our data suggest that this may be the case. When we phosphorylated p47 phox in vitro, we observed quenching of tryptophan fluorescence compared with control samples of p47 phox . As with SDS and arachidonic acid, the treatment of p47 phox required to cause this quenching also results in O 2 . production when tested in a modified cell-free assay system. Two important comparisons can be made between the tryptophan fluorescence quenching seen in phosphorylation versus amphiphile treatment. production. The second point of comparison is the wavelength of maximal fluorescence of treated versus untreated p47 phox . With both amphiphile and phosphorylation treatments, the wavelength of fluorescence maxima did not change significantly from the appropriate control sample. This indicates that any change in the immediate environment of the tryptophan residues caused by a conformational change is similar for both amphiphile and phosphorylation treatments. CD spectroscopy of proteins provides a direct reflection of the secondary structure of the protein (59) and has been used in a number of studies to determine the secondary structure of soluble proteins (e.g. see Refs. 59 and 60). In the present studies, we use CD spectroscopy as a second approach to investigate potential changes in p47 phox conformation induced by amphiphilic agents and to substantiate our fluorescence analyses. The CD spectrum and, therefore, the secondary structure content of p47 phox was not altered by the zwitterionic detergent Zwittergent 3-14, whereas the corresponding CD spectrum of the protein was significantly changed by the addition of the anionic detergent SDS, suggesting that there are indeed changes in the secondary structure of p47 phox in the presence of SDS. This is consistent with our conclusion that both the decrease in intrinsic tryptophan fluorescence and the ability to support NADPH oxidase activation caused by SDS treatment of p47 phox correlate with conformational changes in the protein.
One interesting observation was that the far UV absorption spectrum of p47 phox showed significant flattening after SDS addition. Similar flattening effects have been observed previously in the CD and UV absorption spectra of membrane proteins (39). The flattening of the UV absorption spectrum has been attributed to the high local concentration or aggregation of proteins within a membrane environment. The resulting close packing of the proteins leads to a reduced optical crosssectional area for the chromophores, i.e. the protein backbone peptide bonds, relative to having the same chromophores homogeneously distributed throughout the measured volume. Consequently, the measured intensities of the CD spectrum are lowered in direct proportion to the reduced absorption of the sample. Based on the currently accepted hypothesis that p47 phox changes conformation during NADPH oxidase activation to reveal a sequestered cationic domain containing a p67 phox binding site (p47 phox residues 323-332) (31) and a neighboring SH3 domains (p47 phox residues 151-214 and 227-284) (28,29), it is consistent that analogous SDS-induced conformational changes would increase the tendency of p47 phox to aggregate because of the "sticky" nature of the revealed sites and the absence of NADPH oxidase cofactors to bind to these sites.
In the present studies, we suggest that treatment of p47 phox in vitro with the appropriate dose of amphiphilic agents can induce conformational changes in p47 phox that mimic phosphorylation-induced changes in p47 phox occurring in vivo. It is possible, however, that the interaction of an amphiphile with p47 phox may occur at a different site than where phosphorylation is occurring, but in both cases the conformational change that promotes oxidase assembly may occur at a third site. The methods used here cannot provide precise detail about the nature of these conformational change(s). The tryptophan fluorescence technique indicates that one or more tryptophan residues is affected by this change; however, we do not know whether changes in the tryptophan residues themselves are involved with binding interactions which may result in activation of the oxidase. As mentioned above, p47 phox contains two SH3-binding domains, which may be involved in interactions with other oxidase components; one of these domains contains two tryptophan residues, and the second contains three. Thus, it is possible that the amphiphile-induced unmasking of SH3 domains, as has been described by Sumimoto et al. (28) and Leto et al. (29), could be altering the micro-environment of some of these tryptophan residues, resulting in the observed decreases in tryptophan fluorescence. There are, however, two other tryptophan residues in p47 phox located at sites that are not currently implicated in binding with other oxidase proteins. If the observed changes in tryptophan fluorescence were due only to the latter two tryptophan residues, then changes in conformation in those regions may only be indirectly related to changes at sites of oxidase protein binding interaction. However, in a recent report by Sumimoto et al. (32), a tryptophan in one of the SH3 domains of p47 phox was mutated to arginine, and the mutated p47 phox was unable to support O 2 . production in a cell-free assay (32). This suggests that at least one of these tryptophan residues is important in p47 phox interactions with other oxidase proteins, and warrants further study as to the contribution of that residue to the fluorescence quenching presented here. Such studies could provide useful information about the molecular interactions which form the basis of activation of the neutrophil oxidative burst.