Purification of the Solubilized NADPH:02 Oxidoreductase of Human

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 12, Issue of April 25, pp. 5617-5623, 1988 Printed in U. S. A.

Purification of the Solubilized NADPH:02 Oxidoreductase of Human Neutrophils ISOLATION OF ITS CATALYTICALLY INACTIVE CYTOCHROME b AND FLAVOPROTEIN REDOX CENTERS*

(Received for publication, November 30,1987)

Terrence R. Green$ and Katherine L. Pratt From the Clinical Pathology Service, Veterans Administration Medical Center, and Department of Biochemistu, Oregon Health Sciences University, Portland, Oregon 97201

The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A- Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 "C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with Oz, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.

The enzymology of the NADPH:02 oxidoreductase of human neutrophils has been the subject of intense investigation. Principal dioxygen products of the oxidoreductase appear to be superoxide and, to a lesser extent, hydrogen peroxide (1). It also exhibits intrinsic diaphorase type activity when supplied with appropriate electron acceptors other then O2 such as dichlorophenolindophenol, nitroblue tetrazolium salt (NBT),' ferricyanide, or ferricytochrome c (2-7). Children whose neutrophils lack this oxidoreductase suffer from chronic granulomatous disease (CGD), an inherited disorder associated with recurrent and ultimately fatal infections (8- 10). Oxidoreductase activity is characteristically found firmly associatedwith the plasmalemma of the cell. Catalytic activity is expressed only following cell stimulation. Membrane fractions prepared from resting neutrophils, or stimulated cells derived from CGD donors, lack enzyme activity.

* This work was supported by the Medical Research Foundation of Oregon and the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. 'The abbreviations used are: NBT, nitroblue tetrazolium salt;

DOC, deoxycholate; DTPA, diethylenetriaminepentaacetic acid; CGD, chronic granulomatous disease; PMA, phorbol myristate acetate.

Attempts at purifying and characterizing the oxidoreductase free of the neutrophil membrane with respect to recoverable enzyme activity have not been successful. The enzyme is well-known to be quite unstable once freed of the membrane (11-13). There is evidence, however, that the oxidoreductase is actually a multienzyme complex comprised of discrete redox centers which must work together in affecting the shuttling of electron equivalents given up by NADPH to 0 2 . A cytochrome b of unusually low redox potential is involved in conferring enzyme activity to the oxidoreductase (14). A flavoprotein also appears to be a component of the oxidoreductase (15). In addition, the oxidoreductase requires lipid and a cytosolic factor in order for it to exhibit enzyme activity (16, 17). Hence, the lability of the oxidoreductase freed of the membrane might be accounted for in terms of dissolution and uncoupling of its individual protomeric redox components from one another.

We present here evidence that the solubilized oxidoreductase is a weakly associated protein complex which can be resolved into its component parts including its flavoprotein and cytochrome b components with concomitant loss of both NADPH oxidase and diaphorase activities intrinsic to the intact enzyme complex. Dissociation of the enzymatically active complex occurs under mild conditions such as passage of the solubilized oxidoreductase through molecular gel sieving or concanavalin A affinity columns.

MATERIALS AND METHODS

FAD, ferricytochrome c, NADH, NADPH, ferricyanide, NBT superoxide dismutase, catalase, deoxycholate, Lubrol-PX, diethylenetriaminepentaacetic acid (DTPA), EDTA, sodium dithionite, phorbol myristate acetate (PMA), Tris-HC1, glycine HCl, concanavalin A- Sepharose 4B, and Sephadex G-10 were all obtained from Sigma. Bio- Gel A-1.5m agarose was obtained from Bio-Rad. All other chemicals were of the best grade available.

Isolation of Neutrophils-Buffy-coat and purified neutrophils were isolated from whole blood collected in EDTA as previously described (2). Cells freed of contaminating red blood cells by hypotonic lysis in distilled water were suspended in Hanks' balanced saline solution, pH 7.4, at approximately 4 X lo' cells ml-', stimulated by exposure to PMA (1 pg ml-') for 10 min at room temperature, and harvested by centrifugation at approximately 1,000 X g for 5 min. The cell pellet was lysed by brief sonication in 15% ethylene glycol made up in water and also containing 0.1 mM DTPA adjusted to pH 7.0, then centrifuged for 30 min at 2 "C at 27,000 X g. Oxidoreductase activity was recovered entirely in the 27,000 X g pellet fraction.

Solubilization and Further Purification of the Oxidoreductase-The 27,000 X g pellet fraction was resuspended by sonication to a final protein concentration of approximately 5 mg ml" in solubilization buffer comprised of ice-cold 20 mM glycine-HC1, pH 8.6, made up in 25% glycerol, 0.25% deoxycholate, 0.25% Lubrol-PX, 0.5 mM CaC12, 1 mM MgClz, 0.1 mM DTPA, and 0.5 mM sodium azide. The resuspended pellet fraction was then centrifuged for 60 min at 2 'C at

5617

5618 Purification of the Neutrophil Oxidoreductase

100.000 X g on a type 40 rotor in a model L3-50 Beckman ultracen- trifuge. The 100,000 X g supernatant recovered from this step (solubilized oxidoreductase) served as starting material in further purifying the enzyme complex by affinity and molecular sieving column chromatography (see text).

Enzyme Assays-Four methods of quantitating enzyme activity were employed. These included a determination of superoxide generating activity at 550 nm employing the superoxide dismutase inhib- itable ferricytochrome c assay, NADPH oxidase activity measured at 340 nm, and a determination of NADPH and NADH NBT reductase (diaphorase type) activities (final concentration of NBT, 94 p ~ ) measured at 540 nm. In the latter instance excess superoxide dismutase (150 units) was included in the final reaction mixture to eliminate superoxide-mediated NBT reduction. In addition, NADPH and NADH oxidase activities were measured at 340 nm in the presence of ferricyanide (final concentration, 80 p ~ ) . Except where stated differently in the text, NAD(P)H oxidase activities were routinely assayed with ferricyanide included in the final assay mixtures. The varying oxidase and diaphorase type assays were used at different stages of purification of the oxidoreductase complex as described in the text. All enzyme assays were conducted at room temperature a t pH 7.6 in 32 mM Tris-HC1, 0.25 mM CaC12, 0.16 mM MgCl,, and 154 pM DTPA as previously described on a Shimadzu UV-250/265 double beam spectrophotometer (18). The final concentration of NAD(P)H was 155 p ~ . One milliunit of enzyme activity corresponds to the oxidation of 1 nmol of NAD(P)H/min.

Determination of FAD and Cytochrome b-FAD was measured by fluorescence spectroscopy according to the method of Yagi (19). The excitation fluorescence at 450 nm was routinely monitored with the emission wavelength set at 530 nm, and the difference in fluorescence before and after dithionite addition was then used to calculate FAD content with respect to that observed with authentic FAD of known concentration. Cytochrome b content was estimated by dithionite difference spectroscopy based upon the millimolar absorptivity coef- ficients of 21.6 and 104 at 558 and 424 nm, respectively (12, 20, 21).

Protein Determinations-Protein was determined by the method of Lowry et al. (22). The presence of protein peaks in column effluents was monitored at 280 nm.

RESULTS

Enzyme Activities, FAD, and Cytochrome b Content of the Membrane-bound Oxidoreductase-Table I contrasts enzyme activities of interest pertaining to the oxidoreductase complex in membrane preparations obtained from resting versus phor- bo1 myristate acetate-stimulated cells. The 27,000 X g pellet fractions derived from PMA-stimulated cells clearly show enhanced superoxide generating, NADPH oxidase (measured in the presence and absence of ferricyanide), NADPH and NADH NBT reductase (diaphorase type) activities well above those derived from comparable pellet fractions of resting cells. On the other hand, comparable levels of NADH oxidase

TABLE I Enzyme activities in 27,000 X g membrane fractions of resting versus

PMA-stimulated cells All results expressed as mean of duplicate determinations. Values

are in milliunits/mg" protein except for NBT reductase assays where units are mAbs min"/mg" protein. Excess superoxide dismutase was included in NBT reaction mixtures to ensure rates were not meas- uring superoxide-mediated reduction of NBT. NADH oxidase activity was not measured in the absence of ferricyanide since oxygen has previously been shown to be a very poor electron acceptor of this specific oxidase activity (23). A more detailed discussion of the assays is described under "Materials and Methods."

Enzyme activity Resting Stimulated membrane membrane

Superoxide production 0.38 113 NADPH oxidation

Minus ferricyanide 1.5 63 With ferricyanide 3.4 54

NADPH-NBT reduction 0 59 NADH oxidation

With ferricyanide 304 292 NADH-NBT reduction 75 136

activities were observed with pellet fractions obtained from either resting or PMA-stimulated cells. The FAD content of the membrane preparations exhibiting enhanced oxidoreductase activity averaged 220 f 4 ( n = 4) pmol/mg" protein. Their cytochrome b content was 167 f 33 ( n = 14) pmol/ mg" protein.

Solubilization of the 27,000 X g Pellet Fraction-The recovery of solubilized NADPH oxidase activity measured in the absence and presence of ferricyanide averaged 71 f 12% ( n = 22) and 79 k 21% ( n = 22), respectively, of the starting activity observed in 27,000 x g pellet fractions prepared from PMA-stimulated cells. NADH oxidase activity averaged 111 k 25% ( n = 22) of that in the starting pellet fractions. The slightly higher recovery of solubilized NADH oxidase activity over that in the pellet fractions suggests either latent enzyme activity was released with solubilization or that the assays on the particulate membrane fractions underestimate the true amount of NADH oxidase activity in these fractions.

There was approximately a 10-fold decrease in contamina- tion from myeloperoxidase accompanying solubilization of the oxidoreductase. Figs. 1 and 2 contrast the dithionite difference spectra obtained on enzyme before and after solubilization. Absorption spectra at 558, 530, and 424 nm constitute the reduced minus oxidized spectrum of the cytochrome b component. Those at 636 and 473-474 nm correspond to that of ferrous myeloperoxidase.

The lability of the solubilized oxidoreductase was studied by drawing aliquots of enzyme solutions for assays of residual NADPH oxidase activity at varying intervals after recovery of the 100,000 X g supernatant. Fig. 3 summarizes decay rates seen in typical experiments in which the solubilized oxidoreductase was left standing either in the presence or absence of exogenous FAD (10 WM). The half-life of the solubilized oxidoreductase stored at 2-4 "C in solubilization buffer alone, or supplemented with exogenous FAD, was indistinguishable within experimental error and averaged approximately 30 h.

T

473 I

1 A = 0.0 1

FIG. 1. Dithionite difference spectrum of particulate membrane fraction recovered from PMA-stimulated cells. The membrane fraction was prepared and resuspended in solubilization buffer, pH 8.6, and scanned from 700 to 400 nm as described under "Materials and Methods." Following addition of a few grains of dithionite, a second scan was made recording the reduced minus oxidized spectrum. Numbers indicate the absorption maxima of the difference spectrum.

Purification of the Neutrophil Oxidoreductase 5619

I A = 0.0 1 424

1

FIG. 2. Dithionite difference spectrum of detergent-extracted oxidoreductase (100,000 X g supernatant fraction). Scan conditions were as described in Fig. 1. Numbers indicate absorption maxima of the difference spectrum. For further details see

0 without FAD

A with 10pM FAD

I 1 I I I I 2 3 4 5

Storage Time (Days) FIG. 3. Stability of solubilized oxidoreductase uersus stor-

age time. Enzyme was stored in solubilization buffer, pH 8.6, after recovery in the 100,000 x g supernatant fraction of detergent-extracted membrane preparations rich in oxidoreductase activity. Ali- quots of the solubilized enzyme were then assayed at varying intervals for residual NADPH oxidase activity at 340 nm as described under “Materials and Methods.” Each data point is the average of a single determination for enzyme stored at 2-4 “C in the presence and absence of 10 p~ FAD for the intervals indicated in the drawing.

We were unsuccessful in extending the half-life beyond this interval despite varying the pH of the buffer in which the enzyme was stored, employing different concentrations of detergent, the inclusion of proteinase inhibitors (leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, 1-chloro-3-to- sylamido-7-amino-2 heptanene in the solubilization buffer, and variations in the glycerol content of the final solubilization buffer.

Affinity Column Chromatography-The recovery of oxidase activity from concanavalin A-Sepharose depended upon the extent of resolution of flavoprotein free of the cytochrome b component. With small columns of approximately 10 ml of bed volume, the recovery of NADPH oxidase applied to columns was quantitative. Cytochrome b and flavoprotein were not resolved from one another (data not shown). In scaling up to a packed bed volume of 48 ml, however, the average recovery of NADPH oxidase activity dropped to 20 f 10% (n

= 6). NADH oxidase activity was unaffected by scaling up the column (average recovery 104 -+ 14% (n = 7)). Concomitant with the 80% loss of enzyme activity, we observed significant resolution of the major flavoprotein peak free of the cytochrome b fractions recovered from the concanavalin A-Seph- arose column. This data is shown in Fig. 4. Scaling up another 3-fold in column bed volume led to improved resolution of the cytochrome b and flavoprotein components from one another, and a 98% loss of recoverable NADPH oxidase activity. In the latter instance (cf. Fig. 5 ) minor fractions of unbound cytochrome b and flavin were completely resolved from major peaks constituting the bulk (e.g. approximately 75%) of the cytochrome b and flavin applied to the column. What little NADPH oxidase activity was recovered eluted from the column in the overlap region between the flavoprotein and cytochrome b peaks (cf. approximately fraction 32 in Fig. 5 ) . NADH oxidase activity was recovered quantitatively. NADH- NBT reductase activity, which co-eluted with the NADPH oxidase, was recovered at less than 10% of that applied to the column.

We considered the possibility that the losses in oxidoreductase activity might be attributable to its lability in solubilized form. However, the time elapsed between application of the solubilized oxidoreductase to the affinity columns and recovery of enzyme activities never exceeded approximately 3 h, an

i 0

2 4 6 8 10 12 22 24 26 28 30

Fraction Number

FIG. 4. Concanavalin A-Sepharose affinity chromatography of solubilized oxidoreductase. Detergent extracted 100,000 X g supernatant recovered from oxidoreductase-rich membrane preparations in solubilization buffer, pH 8.6, was applied to a concanavalin A-Sepharose column (packed bed volume, 48 ml) equilibrated in the same buffer at pH 8.6. The column effluent was assayed for protein (-), cytochrome b (W), flavin (A-A), NADH (o”--o), and NADPH (A-A) oxidase activities as described under “Mate- rials and Methods.” Each fraction = 3.3 ml. Solubilization buffer supplemented with methylmannoside (0.5 M) was applied to the column at fraction 21 as indicated by the arrow to displace protein bound tightly to the concanavalin ligand. No enzyme activity was recovered in the tightly bound fraction eluting after addition of methylmannoside to the column.

Purification of the Neutrophil Oxidoreductase

3 c amounts in the void volume peak but principally eluted in the

of eluted enzyme activities recovered from a typical Sephadex G-10 column as opposed to that observed with Bio-Gel A- 1.5m-agarose column (void volumes of each column were adjusted to approximately 75 ml).

Since the loss of oxidoreductase activity was most notable on the Bio-Gel A-1.5m-agarose column, the distribution of cytochrome b and flavoprotein eluting from this column was also determined and is shown in Fig. 7. Approximately 25% of the total flavin and cytochrome b recovered from the column eluted with the void volume corresponding to the fractions exhibiting NADPH oxidase activity (cf. Fig. 6). The remainder of the cytochrome b was resolved free of flavoprotein (cf. Fig. 7). A major flavoprotein peak recovered from the column (approximately 70% of the total flavin applied to the column) eluted as a single symmetrical peak of apparently

W lower molecular weight than that of the cytochrome b frac- 8 1 tions. Its elution point on the column (fraction 51, Fig. 7) - suggests a molecular weight of approximately 51,000 based

E 200,000 molecular weight range. Fig. 6 summarizes the pattern

40

-

5620

2

10 30 40 60

Fraction Number FIG. 5. High resolution concanavalin A-Sepharose affinity

chromatography of solubilized oxidoreductase. Detergent extracted 100,000 X g supernatant was applied to a scaled-up concanavalin A-Sepharose column (packed bed volume, 165 ml) as in Fig. 4. The column effluent was assayed for cytochrome b (lower panel, u), flavin (lower panel, A-A), NADH oxidase (upper panel, M), NADPH oxidase (upperpanel, A-A), and NADH-NBT diaphorase (upper panel, W) activities as described in the text. The scale in the upper panel for NADH oxidase activity is compressed by factor of 100 (peak enzyme activity in fraction 31 = 345 milliunits/ ml-l). FU = relative fluorescence units (chart full scale = 100 FU). Peak NADH oxidase eluted in fraction 31; peak NADPH oxidase and NADH-NBT diaphorase activities co-eluted in fraction 32. The principal cytochrome b and flavin peaks eluted in fractions 27-29 and fractions 33-34, respectively. Each fraction = 4.2 ml.

interval of time far shorter than the half-life of the solubilized oxidoreductase (cf. Fig. 3). Furthermore, we were able to confirm that enzyme stored in the cold room for the duration of the chromatography experiments, but not passed through the affinity columns, had not lost any significant activity over this interval of time.

Molecular Exclusion Chromatography of the Solubilized Ox- idoreductase-The relationship between resolution of the flavoprotein and cytochrome b components of the oxidoreductase and loss of apparent catalytic activities was further investigated by passage of the solubilized oxidoreductase through gel sieving columns of varying exclusion limits. Upon passage through a Sephadex G-10 column (molecular weight exclusion limit, lO,OOO), NADPH oxidase activity was recovered in quantitative yield. This compared with 63% recovery with passage through a Sephadex G-100 column (exclusion limit, 100,000), and 20% recovery on a Bio-Gel A-1.5m-agarose column (exclusion limit, 1.5 x lo6). In each instance, as in the case of the concanavalin A affinity columns, the transit time of applied enzyme through the column was too short to explain the loss of activity in terms of decay of applied enzyme. NADPH oxidase activity was found solely in the void volume peaks of the gel sieving columns. With Bio-Gel A- 1.5m-agarose, NADH oxidase activity was found in small

upon calibration of the column with proteins- of known molecular weight. Proteins used to calibrate the column were trypsin (23,800), hemoglobin (64,500), alkaline phosphatase (SO,OOO), glucose oxidase (150,000), and the NADH diaphorase

10 20 30 40 50

Fraction Number FIG. 6. Molecular sieving chromatography of solubilized

oxidoreductase. a, protein (-), NADPH (A-A), and NADH (U) oxidase activities recovered upon passage of the solubilized 100,000 X g supernatant fraction rich in oxidoreductase activity through a Bio-Gel A-1.5m-agarose column equilibrated in solubilization buffer, pH 8.6 (void volume, approximately 75 ml). b, distribution of same enzyme activities and protein upon passage of comparable solubilized oxidoreductase through a similarly equilibrated column of Sephadex G-10 (void volume, approximately 75 ml). Recovery of NADPH oxidase in a was 20%; recovery of NADPH oxidase in b was 100%. NADH oxidase activity was recovered in quantitative yield from both columns. Each fraction = 3.4 ml. Excluded protein eluted in fraction 22 for both columns. For further details see text.


t 0

I I 2.0 - I i 8

q 1.0- s *"

1.4- J E

d 1.2 - 8

* 0.8 - 0.6 - 0.4 - 0.2 -

I

t 1 5 0

1 0 2 0 3 0 4 0 5 0 6 0 ~ 0

Fraction Number FIG. 7. Resolution of cytochrome b and flavoprotein com-

ponents of oxidoreductase upon passage of solubilized enzyme through Bio-Gel A-1.6m-agarose. Column conditions were as in Fig. 6. The resolved flavoprotein peak (fraction 51) was recovered in an elution volume of 152 ml (void volume = 75 ml). Each fraction = 3.2 mi. FU = relative fluorescence units, The molecular weight of this peak corresponds to a molecular weight of 51,000 based upon calibration of the column with known protein standards (see text).

(204,000) previously characterized in this laboratory (23). No NADPH oxidase activity was detectable in any of the

fractions recovered from the Bio-Gel A-1.5m-agarose column except in the void volume where a minor amount of cytochrome 6 and flavoprotein co-eluted. Neither the cytochrome 6 nor the flavoprotein peaks by themselves exhibited any NAD(P)H oxidase activities. They were also devoid of NAD(P)H-NBT reductase activities.

Fig. 8 shows the fluorescence excitation spectrum of fraction 51 recovered from the Bio-Gel A-1.5m-agarose column. Characteristic fluorescence peaks for FAD are evident at 375 and 452 nm with a slight shoulder peak at 465 nm.

DISCUSSION

Attached to the membrane the oxidoreductase exhibits 02- generating activity in the range of 50 to several hundred milliunits/mg-l protein depending upon the source of neutrophils (e.g. guinea pig, pig, bovine, or human), the manner in which the cells are disrupted following activation (e.g. cavi- tation, enucleation, sonication, etc.), and subsequent process- ing of the cell lysates (e.g. differential centrifugation and collection of pellet fractions versus density gradient centrifugation such as with Percoll) (4, 23-29). Despite the relative ease of obtaining crude enzyme preparations, solubilization and purification of the oxidoreductase has proven far more difficult. The cytochrome 6 component has been purified free of the membrane, for example, but is completely devoid of enzyme activity (20, 21, 30, 31). Doussiere and Vignais (12) attempted to purify the oxidoreductase, but they recovered no more than approximately 1% of the starting activity in their crude enzyme extracts after subjecting the solubilized oxidoreductase to ion exchange and gel-sieving chromatography. Babior and co-workers (13, 32) have also presented data on purification of the oxidoreductase but similarly achieved an actual recovery of less than 1 % of the initial enzyme activity present in their crude enzyme extracts. Gabig and Lefker (9,

465

FIG. 8. Fluorescence excitation spectrum of Bio-Gel A- 1.Sm-agarose resolved flavoprotein. Fraction 51 (cf. Fig. 7) was scanned from 300 to 500 nm at a slit width of 10 mm on a Perkin- Elmer model 4 Fluorometer with the emission wavelength held con- stant at 530 nm. Fluorescent scans were conducted in solubilization buffer, pH 8.6. Values noted in the drawing indicate the wavelengths at which maximum fluorescence excitation occurred in the sample.

18) lost virtually all O,-generating activity upon solubilizing the oxidoreductase in cholate. They observed partitioning of the flavoprotein and cytochrome b components into separate fractions with centrifugation and proposed that the separation of these two redox centers from one another accounted for loss of 0,-generating activity.

Evidence that cytochrome 6 is a component of the oxidoreductase complex is compelling and includes extensive studies documenting the absence of this component in membrane preparations of neutrophils derived from most patients with the X-linked form of CGD (&lo), detailed studies documenting NADPH-dependent reduction of the cytochrome 6 component under anaerobic conditions, and its spontaneous oxidation with readmission of O2 to reaction mixtures ( l l ) , and cloning of the gene associated with the X-linked form of CGD and documentation that a portion of this gene codes for a 91,000 molecular weight glycopeptide-anchoring protein which binds the cytochrome 6 component to the cell membrane (33). Approximately 90% of the total cytochrome b found in the granular membrane fraction rich in oxidoreductase activity appears to be of very low redox potential (e.g.

5622 Purification of the Neutrophil Oxidoreductase

approximately -240 mV), the form believed to be involved in catalyzing formation of Os- (20, 34). Hence, it is reasonable to conclude that most of the cytochrome b resolved into discrete fractions on the concanavalin A and Bio-Gel A-1.5m- agarose columns is involved in the oxidoreductase complex, that the losses in enzyme activity seen with progressively higher degrees of resolution of this component on the columns reflects dissociation of the oxidoreductase and resolution of its protomers free of the cytochrome b component, and that with uncoupling from the cytochrome b component, that the protomers lack the capacity to affect oxidation of NADPH and concomitant reduction of 02.

The 51,000 molecular weight flavoprotein recovered from the concanavalin A-Sepharose and Bio-Gel A-1.5m-agarose columns is also very likely a protomeric component of the oxidoreductase complex for the following reasons. First, Kak- inuma et al. (15) have established by EPR spectroscopy that a flavoprotein is involved in the oxidoreductase complex. Second, and of particular importance, a comparison of the level of FAD found in granular membrane preparations of normal donors to those derived from donors with rare flavin- deficient forms of CGD indicates that somewhere between 60-80% of the total FAD found in the granular membrane preparations of normal donors is associated with the oxidoreductase. For example, Umei et al. (35) reported the normal FAD content of their granular membrane preparations to be 280 pmol/mg" of protein. In surveying for FAD-deficient forms of CGD, they identified five patients whose neutrophil membranes contained from 20 to 40% of the normal level. Ohno et al. (36) identified four CGD patients with levels of FAD ranging from 23 to 32% normal. Bohler et al. (37) also found four CGD patients with FAD levels ranging from 24 to 35% of the normal range of healthy donors. Borregaard and Tauber (38) have presented data on two flavin deficient CGD patients whose neutrophil membranes contained approximately 20% of the normal level. Lastly, Gabig and Lefker (24) reported finding two CGD patients whose neutrophil membranes were devoid of FAD. Hence, from these genetic studies we conclude that the bulk of the FAD found in the membrane fractions rich in oxidoreductase activity must be associated with the oxidoreductase complex. It thus follows from the distribution data on the flavoproteins recovered from the solubilized oxidoreductase (cf. Figs. 5 and 7 ) that the 51,000 molecular weight flavoprotein, which contains roughly 70% of the total FAD found in granular membrane preparations, is the part of the oxidoreductase complex missing in CGD patients with the flavin deficiency.

The fact that NADH oxidase activity resolves cleanly from the 51,000 molecular weight flavoprotein peak (cf. Figs. 6 and 7) , the quantitative recoveries of this enzyme activity despite losses in NADPH oxidase and NBT reductase activities, and lastly the fact that this enzyme is not able to catalyze the oxidation of NADPH as shown in an earlier study of its properties from this laboratory (23), indicates that it is not a component of the oxidoreductase. Since all other protein fractions including the major flavoprotein and cytochrome b peaks were devoid of enzyme activity, it follows that the resolved parts of the oxidoreductase complex recovered from the Bio-Gel A-1.5m-agarose column are devoid of measurable catalytic activity. Hence, with dissociation or resolution of the individual redox centers of the oxidoreductase, free of one another, uncoupling occurs whereby electron equivalents supplied by NADPH are no longer transferable to either O2 or alternate electron acceptors capable of catalyzing transfer steps in the assembled enzyme complex.

This conclusion is in opposition to recent data presented

elsewhere regarding the nature of the flavoprotein component of the oxidoreductase complex. For example, Kakinuma et al. (6) have reported separating a FAD enzyme exhibiting NADPH-dependent NBT diaphorase activity free of the cytochrome b component by isoelectric focusing. Its molecular weight was reported to be 67,000. Sakane et al. (7 ) have also reported that they have succeeded in purifying a NADPH- cytochrome c reductase exhibiting a molecular weight of 80,000 from neutrophil membranes free of the cytochrome b component, and that this latter enzyme when mixed together with the purified cytochrome b component and phosphatidyl- choline-catalyzed NADPH-dependent 02-generating activity. Since the recovery of flavoprotein in terms of total flavin present in their respective starting membrane preparations was not reported, it is difficult to ascertain at this time the relationship of these flavoproteins to that proposed in this study to be a component of the oxidoreductase. In addition, because the enzymes described by these two groups were not prepared from human neutrophils, species differences might explain the different findings between our observations on the properties of the flavoprotein component. The molecular weight of our flavoprotein is clearly smaller than either of the two described above, and it is also quite distinct in lacking any trace of measurable NADPH oxidase or diaphorase type activities when resolved free of the cytochrome b component.

The observation that the resolved flavoprotein is completely deficient in catalytic activity, including diaphorase type activities, is important. It was initially anticipated that the flavoprotein component might exhibit by itself intrinsic diaphorase activity, and thus with a suitable electron acceptor, its purification could be followed. The fact that diaphorase activity was lost concomitant with resolution of the flavoprotein free of the cytochrome b component suggests that the NADPH-binding site is either formed by association of the cytochrome b component with the 51,000 molecular weight flavoprotein component, or possibly a third protein containing the NADPH binding site is also involved in the oxidoreductase complex. If the latter case is true, then it must also have been resolved free of the flavoprotein on the concanavalin A- Sepharose and Bio-Gel A-1.5m-agarose columns if we are to account for the loss of all forms of enzyme activities noted in this study. In this respect, it is of considerable interest that Umei et al. (36) have recently succeeded in radiolabeling the NADPH-binding site of the oxidoreductase with 2',3'-dialde- hyde NADPH and sodium cyan~boro[~H]hydride. They found upon sodium dodecyl sulfate-gel electrophoresis that the NADPH-binding site exhibited a molecular weight of 66,000. They noted that the protein labeled with NADPH was not diminished in CGD patients deficient in flavoprotein. This may suggest that the protein responsible for binding NADPH is distinct from the flavoprotein and thus that the oxidoreductase complex is comprised of at least three distinct protomers, a cytochrome b, a flavoprotein of approximately 51,000 in molecular weight, and a third protein involved in facilitat- ing binding of NADPH to the oxidoreductase and transfer of its electron equivalents to the flavoprotein component.

It is possible that the third protein component of the oxidoreductase is the cytosolic factor found missing in certain patients with the autosomal recessive form of CGD (39). Bromberg et al. (40) have presented preliminary evidence suggesting that the cytosolic factor contains the NADPH- binding site. Curnutte and co-workers (41) have shown that the latter protein component subjected to gel-sieving chromatography in nondenaturing buffer elutes as a major peak with a molecular weight of approximately 250,000 and as a minor peak at approximately 40,000. We have not attempted


reconstitution experiments to ascertain whether protein in the 250,000 molecular weight range recovered from the Bio- Gel A-1.5m-agarose column in combination with the cytochrome b and 51,000 molecular weight flavoprotein component exhibits oxidoreductase activity. Reconstitution experiments of this type are now clearly feasible in ascertaining the role of the cytosolic factor in relation to that of the cytochrome b and 51,000 molecular weight flavoprotein components and are currently in progress.

Acknowledgment-We thank Carrie McKinnon for technical as- sistance in the preparation of membrane-bound oxidoreductase used as starting material in this studv.

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