AND IMMUNITY, May 1978, p. 398-405 0019-9567/78/0020-0398$02.00/0 Copyright © 1978 American Society for Microbiology
INFECTION
Vol. 20, No. 2 Printed in U.S.A.
Allosteric Transformation of Reduced Nicotinamide Adenine Dinucleotide (Phosphate) Oxidase Induced by Phagocytosis in Human Polymorphonuclear Leukocytes LAWRENCE R.
DECHATELET,'2*
PAMELA S. SHIRLEY,' LINDA C. McPHAIL,' DAVID B. AND GEORGE J. DOELLGAST'
IVERSON,'
Departments of Biochemistry' and Medicine,2 Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103
Received for publication 17 October 1977
We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or ,8-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.
Phagocytosis by polymorphonuclear leukocytes (PMNL) is accompanied by marked alterations in the oxidative metabolism of the cell, collectively referred to as the "respiratory burst" (8). The initiation of this respiratory burst is generally conceded to be caused by activation of a reduced pyridine nucleotide oxidase, which catalyzes the reaction between the reduced nucleotide and molecular oxygen to yield, ultimately, the oxidized form of the nucleotide and hydrogen peroxide. The nature of the substrate for the reaction is still a matter of some controversy; many workers feel the enzyme is specific for reduced nicotinamide adenine dinucleotide (NADH) (2, 4, 5, 25, 26), whereas others have presented evidence that suggests reduced NAD phosphate (NADPH) is the critical substrate (14, 21-24). We have recently adapted sensitive assay techniques for these oxidative activities
(11, 17, 20) and presented some evidence that suggests that a single enzyme might be responsible for oxidation of either substrate in the human PMNL, although it appears to be substantially more active toward low concentrations of NADPH than of NADH (17). If the physiological substrate is controversial, still less is known about the activation of the enzyme by phagocytosis. Patriarca et al., using guinea pig cells, have reported that the Km of NADPH oxidase decreases tenfold after initiation of phagocytosis, whereas the Vmax increases fourfold, and suggested this as the mechanism for activation of the respiratory burst (22). Similar alterations were reported by the same group for rabbit PMNL as well as for human PMNL (24). These assays, however, were performed in the presence of added Mn2"; Curnutte et al. have demonstrated that this metal ion introduces a 398
VOL. 20, 1978
NAD(P)H OXIDASE IN PMNL
399
major artifact in the assay by catalyzing a non- Each sample was homogenized for a total of 5 min in enzymatic oxidation of the reduced nucleotides 1-min intervals with cooling in between. The whole (6). Accordingly, the physiological significance homogenate was initially centrifuged at 500 x g for 10 min in the cold to remove unbroken cells, nuclei, large of these observations is not certain. debris, and zymosan. The 500 x g supernatant was In the present paper, we report the effect of then centrifuged at 27,000 x g for 15 min, and the substrate concentration on NAD(P)H oxidase(s) resulting granule pellet was resuspended in 0.34 M in a granule preparation derived from normal sucrose with a Dounce homogenizer. The protein conand chronic granulomatous disease (CGD) leu- centration of the resulting suspension was determined kocytes under both resting and phagocytizing by the method of Lowry et al. (19), and all samples conditions. We observed that the NAD(P)H ox- were diluted to a final concentration of 1 mg/ml with idase in normal resting cells appears to be an 0.34 M sucrose for ease in comparing day-to-day reallosteric enzyme with very low activity at phys- sults with different preparations. For the sake of simplicity, this preparation is reiological NAD(P)H concentrations. After phagferred to as the "granule fraction," although its comocytosis, there was a conversion to a form dis- position heterogeneous. In addition to the true playing hyperbolic kinetics with significantly granules isofquite the cell, the preparation contains substanhigher activity at physiological NAD(P)H con- tial amounts of plasma membrane, as evidenced by centrations. This conversion did not take place the presence of Mg2e-adenosine triphosphatase, a after phagocytosis by leukocytes from patients marker enzyme for the plasma membrane in human with CGD. We suggest that this conversion of PMNL (13). Granule fractions derived from resting the oxidative activity is essential for the effective and phagocytizing cells appeared comparable. There was no significant difference between the two prepakilling of phagocytized bacteria. rations in either specific or total activities of Mg2+adenosine triphosphatase, ,8-glucuronidase, acid phosMATERIALS AND METHODS Preparation of leukocyte granule fraction. Heparinized venous blood was obtained from normal healthy volunteers and from nine patients with documented CGD of childhood. A description of the patients studied has previously been published (21); two had the classic X-linked recessive form of the disease, whereas seven had the autosomal recessive form. The erythrocytes were sedimented with Plasmagel (HTI Corp., Buffalo, N.Y.), and the leukocytes were isolated as previously described (9). The leukocytes were suspended to a final concentration of 1.5 x 10" PMNL/ml. These preparations routinely contained 70 to 85% neutrophils, 1 to 7% eosinophils, and 10 to 20% lymphocytes and monocytes. Zymosan (ICN, Nutritional Biochemicals Division, International Chemical and Nuclear Corp., Cleveland, Ohio) was suspended in phosphate-buffered saline at a concentration of 50 mg/ml. Pooled human serum, 2 volumes, was added to 1 volume of the zymosan suspension, and the mixture was incubated for 30 min at 37°C with gentle shaking. At the end of the incubation, the opsonized zymosan was sedimented by centrifugation at 17,000 x g for 10 min. The supernatant was discarded, and the zymosan was resuspended in a volume of phosphate-buffered saline equal to that of the original supernatant. A 1-ml amount of cell suspension was incubated with 2 ml of phosphate-buffered saline (resting cells) or with 2 ml of the opsonized zymosan suspension (phagocytizing cells). The phosphate-buffered saline and zymosan were first equilibrated to 37°C, the cells were added, and the mixture was incubated for 3 min at 37°C. The incubation was stopped by the addition of 3 ml of cold 0.68 M sucrose and immediate immersion of the samples in an ice bath. The cell suspensions were then homogenized in the cold to >90% breakage in a Potter-Elvehjem homogenizer with a motordriven Teflon pestle run at 12,000 rpm (Tri-R Stirrer model S-63C, Tri-R Instruments Inc., Jamaica, N.Y.).
phatase, alkaline phosphatase, or lysozyme. There was no evidence of zymosan particles in this fraction as monitored by phase microscopy. Furthermore, when cells were incubated with zymosan labeled with 1251, less than 2% of the label was recovered in the isolated granule fraction. This was similar to the amount found in the soluble fraction (27,000 x g supematant). To obtain reproducible results, it appears necessary to use the granule fraction immediately after preparation. Freezing and thawing or even prolonged storage in the cold seems to activate the resting granule fraction so that differences between resting and phagocytizing fractions are minimized. Isotopic assay for NADPH oxidase. The isotopic assay for NADPH oxidase has been described in detail previously (11, 20). A standard curve using known amounts of NADP+ was run in parallel with each experiment to allow expression of specific activity of the enzyme in terms of nanomoles of NADP+ produced/30 min per 0.10 mg of granule protein. Fluorometric assay for NAD(P)H oxidases. The fluorometric assay was performed as previously described (17), with several modifications. As with the isotopic procedure, this uses a two-step incubation. The first step consists of a 1.0-ml incubation volume containing 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0-2.0 mM KCN, NADH, or NADPH at the indicated concentration and 0.10 mg of the appropriate granule fraction. The reduced pyridine nucleotides were of fluorometric grade obtained from Boehringer Mannheim, Indianapolis, Ind. Other sources of substrate were quite unsatisfactory in that they contained appreciable quantities of fluorescent contaminants which yielded very high blank values and tended to mask the ,activity of the oxidase. MES was substituted for the phosphate buffer previously used because it has a much greater buffer capacity at the desired pH. Previous studies in our own laboratory and others used 0.1 M phosphate buffer, pH 5.5 (14, 21-23). When we measured the actual pH of a typical
1
400
DECHATELET ET AL.
INFECT. IMMUN.
incubation mixture, it was very close to 6.0, presumably due to the strongly basic nature of the KCN. Accordingly, we turned to MES buffer at pH 6.0, which does not alter its pH in the complete assay system. The reaction was started by the addition of substrate. After 30 min it was stopped with 0.10 ml of 2.2 HCI and then neutralized with 0.10 ml of 2.2 N NaOH. Precipitated protein was removed by centrifugation. Controls were always run in parallel in which the granule fraction was omitted in order to account for the spontaneous (nonenzymatic) oxidation of the nucleotides. A 0.10-ml sample of the supernatant from this incubation was added to 0.15 ml of 10 N NaOH and allowed to incubate for 60 min at room temperature. To the remainder of the first incubation was added 0.05 U of NADase (Worthington Biochemicals Corp., Freehold, N.J.) in 1 M potassium phosphate buffer (pH 7.5), and the tube was reincubated for 2 h at 37°C. This procedure was experimentally determined to completely destroy 80 nmol of either NAD or NADP such that they would no longer form a fluorescent product. After treatment with NADase, a 0.10-ml sample of the solution was again treated with strong base. After incubation in 6 N NaOH, the samples (before and after NADase treatment) were diluted with 1.6 ml of water, and the fluorescence was measured in a Farrand A-4 fluorometer equipped with a 365-nm primary filter and a 448-nm secondary filter. The difference between the two fluorescence values was taken as the amount of NAD(P) formed in the first incubation. Standards were run in each experiment and subjected to the same treatment as all of the other samples. Activities are generally expressed as nanomoles of NAD(P)+ produced/30 min per 0.10 mg of protein. Other enzyme assays. The activities of two other granule-associated enzymes were determined as a function of substrate concentration. Assay conditions were exactly the same as those used for measurement of the oxidase activities, i.e., 0.1 M MES buffer (pH 6.0), 2.0 mM KCN, and 0.10 mg of granule fraction derived from resting or phagocytizing cells. Acid phosphatase activity was determined at varying levels of
t20 \
p-nitrophenylphosphate under these conditions. After a 30-min incubation, the reaction was stopped by the addition of 10 ml of 0.1 N NaOH, and the absorbance at 410 nm was measured on a Beckman DU spectrophotometer. A standard curve using known amounts of p-nitrophenol was used to calculate specific activities in terms of micromoles ofp-nitrophenol formed/30 min per 0.10 mg of protein. ,8-Glucuronidase activity was determined in a similar fashion, using varying concentrations of phenolphthalein glucuronide as substrate. Because of the very low activity of this enzyme under the incubation conditions, it was necessary to use a prolonged incubation time (20 h). At the end of this time the reaction was stopped by the addition of 3.0 ml of 0.34 M glycine-NaOH buffer (pH 10.0), and the absorbance was measured at 540 nm. A standard curve of known concentrations of phenolphthalein was used to calculate the specific activity as micromoles of phenolphthalein formed/20 h per 0.10 mg of protein.
RESULTS Figure 1 shows the effect of varying pH on the oxidation of NADPH determined by the isotopic assay procedure. This experiment was run with 0.17 mM NADPH in the presence of 2.0 mM cyanide. The normal phagocytizing granule fraction shows substantial activity above the spontaneous control activity, with a pH optimum very close to 6.0. A buffer effect is also noticed; MES shows greater activity than acetate, whereas phosphate demonstrates the gre:.test activity. In contrast, the phagocytizing granule fraction from a patient with CGD fails to show activity above the spontaneous oxidation over the entire pH range studied. It should be emphasized that the pH values listed were experimentally determined on the actual incubation mixture and are not simply the nominal pH of the buffer used. A comparison of the pH of the solutions at the beginning and end of the 30-min incubation showed that the pH did not change CGO GRAN Fx
NORM. GRAN Fx
CONTROL
l5
1,
INFECTION
Vol. 20, No. 2 Printed in U.S.A.
Allosteric Transformation of Reduced Nicotinamide Adenine Dinucleotide (Phosphate) Oxidase Induced by Phagocytosis in Human Polymorphonuclear Leukocytes LAWRENCE R.
DECHATELET,'2*
PAMELA S. SHIRLEY,' LINDA C. McPHAIL,' DAVID B. AND GEORGE J. DOELLGAST'
IVERSON,'
Departments of Biochemistry' and Medicine,2 Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103
Received for publication 17 October 1977
We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or ,8-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.
Phagocytosis by polymorphonuclear leukocytes (PMNL) is accompanied by marked alterations in the oxidative metabolism of the cell, collectively referred to as the "respiratory burst" (8). The initiation of this respiratory burst is generally conceded to be caused by activation of a reduced pyridine nucleotide oxidase, which catalyzes the reaction between the reduced nucleotide and molecular oxygen to yield, ultimately, the oxidized form of the nucleotide and hydrogen peroxide. The nature of the substrate for the reaction is still a matter of some controversy; many workers feel the enzyme is specific for reduced nicotinamide adenine dinucleotide (NADH) (2, 4, 5, 25, 26), whereas others have presented evidence that suggests reduced NAD phosphate (NADPH) is the critical substrate (14, 21-24). We have recently adapted sensitive assay techniques for these oxidative activities
(11, 17, 20) and presented some evidence that suggests that a single enzyme might be responsible for oxidation of either substrate in the human PMNL, although it appears to be substantially more active toward low concentrations of NADPH than of NADH (17). If the physiological substrate is controversial, still less is known about the activation of the enzyme by phagocytosis. Patriarca et al., using guinea pig cells, have reported that the Km of NADPH oxidase decreases tenfold after initiation of phagocytosis, whereas the Vmax increases fourfold, and suggested this as the mechanism for activation of the respiratory burst (22). Similar alterations were reported by the same group for rabbit PMNL as well as for human PMNL (24). These assays, however, were performed in the presence of added Mn2"; Curnutte et al. have demonstrated that this metal ion introduces a 398
VOL. 20, 1978
NAD(P)H OXIDASE IN PMNL
399
major artifact in the assay by catalyzing a non- Each sample was homogenized for a total of 5 min in enzymatic oxidation of the reduced nucleotides 1-min intervals with cooling in between. The whole (6). Accordingly, the physiological significance homogenate was initially centrifuged at 500 x g for 10 min in the cold to remove unbroken cells, nuclei, large of these observations is not certain. debris, and zymosan. The 500 x g supernatant was In the present paper, we report the effect of then centrifuged at 27,000 x g for 15 min, and the substrate concentration on NAD(P)H oxidase(s) resulting granule pellet was resuspended in 0.34 M in a granule preparation derived from normal sucrose with a Dounce homogenizer. The protein conand chronic granulomatous disease (CGD) leu- centration of the resulting suspension was determined kocytes under both resting and phagocytizing by the method of Lowry et al. (19), and all samples conditions. We observed that the NAD(P)H ox- were diluted to a final concentration of 1 mg/ml with idase in normal resting cells appears to be an 0.34 M sucrose for ease in comparing day-to-day reallosteric enzyme with very low activity at phys- sults with different preparations. For the sake of simplicity, this preparation is reiological NAD(P)H concentrations. After phagferred to as the "granule fraction," although its comocytosis, there was a conversion to a form dis- position heterogeneous. In addition to the true playing hyperbolic kinetics with significantly granules isofquite the cell, the preparation contains substanhigher activity at physiological NAD(P)H con- tial amounts of plasma membrane, as evidenced by centrations. This conversion did not take place the presence of Mg2e-adenosine triphosphatase, a after phagocytosis by leukocytes from patients marker enzyme for the plasma membrane in human with CGD. We suggest that this conversion of PMNL (13). Granule fractions derived from resting the oxidative activity is essential for the effective and phagocytizing cells appeared comparable. There was no significant difference between the two prepakilling of phagocytized bacteria. rations in either specific or total activities of Mg2+adenosine triphosphatase, ,8-glucuronidase, acid phosMATERIALS AND METHODS Preparation of leukocyte granule fraction. Heparinized venous blood was obtained from normal healthy volunteers and from nine patients with documented CGD of childhood. A description of the patients studied has previously been published (21); two had the classic X-linked recessive form of the disease, whereas seven had the autosomal recessive form. The erythrocytes were sedimented with Plasmagel (HTI Corp., Buffalo, N.Y.), and the leukocytes were isolated as previously described (9). The leukocytes were suspended to a final concentration of 1.5 x 10" PMNL/ml. These preparations routinely contained 70 to 85% neutrophils, 1 to 7% eosinophils, and 10 to 20% lymphocytes and monocytes. Zymosan (ICN, Nutritional Biochemicals Division, International Chemical and Nuclear Corp., Cleveland, Ohio) was suspended in phosphate-buffered saline at a concentration of 50 mg/ml. Pooled human serum, 2 volumes, was added to 1 volume of the zymosan suspension, and the mixture was incubated for 30 min at 37°C with gentle shaking. At the end of the incubation, the opsonized zymosan was sedimented by centrifugation at 17,000 x g for 10 min. The supernatant was discarded, and the zymosan was resuspended in a volume of phosphate-buffered saline equal to that of the original supernatant. A 1-ml amount of cell suspension was incubated with 2 ml of phosphate-buffered saline (resting cells) or with 2 ml of the opsonized zymosan suspension (phagocytizing cells). The phosphate-buffered saline and zymosan were first equilibrated to 37°C, the cells were added, and the mixture was incubated for 3 min at 37°C. The incubation was stopped by the addition of 3 ml of cold 0.68 M sucrose and immediate immersion of the samples in an ice bath. The cell suspensions were then homogenized in the cold to >90% breakage in a Potter-Elvehjem homogenizer with a motordriven Teflon pestle run at 12,000 rpm (Tri-R Stirrer model S-63C, Tri-R Instruments Inc., Jamaica, N.Y.).
phatase, alkaline phosphatase, or lysozyme. There was no evidence of zymosan particles in this fraction as monitored by phase microscopy. Furthermore, when cells were incubated with zymosan labeled with 1251, less than 2% of the label was recovered in the isolated granule fraction. This was similar to the amount found in the soluble fraction (27,000 x g supematant). To obtain reproducible results, it appears necessary to use the granule fraction immediately after preparation. Freezing and thawing or even prolonged storage in the cold seems to activate the resting granule fraction so that differences between resting and phagocytizing fractions are minimized. Isotopic assay for NADPH oxidase. The isotopic assay for NADPH oxidase has been described in detail previously (11, 20). A standard curve using known amounts of NADP+ was run in parallel with each experiment to allow expression of specific activity of the enzyme in terms of nanomoles of NADP+ produced/30 min per 0.10 mg of granule protein. Fluorometric assay for NAD(P)H oxidases. The fluorometric assay was performed as previously described (17), with several modifications. As with the isotopic procedure, this uses a two-step incubation. The first step consists of a 1.0-ml incubation volume containing 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0-2.0 mM KCN, NADH, or NADPH at the indicated concentration and 0.10 mg of the appropriate granule fraction. The reduced pyridine nucleotides were of fluorometric grade obtained from Boehringer Mannheim, Indianapolis, Ind. Other sources of substrate were quite unsatisfactory in that they contained appreciable quantities of fluorescent contaminants which yielded very high blank values and tended to mask the ,activity of the oxidase. MES was substituted for the phosphate buffer previously used because it has a much greater buffer capacity at the desired pH. Previous studies in our own laboratory and others used 0.1 M phosphate buffer, pH 5.5 (14, 21-23). When we measured the actual pH of a typical
1
400
DECHATELET ET AL.
INFECT. IMMUN.
incubation mixture, it was very close to 6.0, presumably due to the strongly basic nature of the KCN. Accordingly, we turned to MES buffer at pH 6.0, which does not alter its pH in the complete assay system. The reaction was started by the addition of substrate. After 30 min it was stopped with 0.10 ml of 2.2 HCI and then neutralized with 0.10 ml of 2.2 N NaOH. Precipitated protein was removed by centrifugation. Controls were always run in parallel in which the granule fraction was omitted in order to account for the spontaneous (nonenzymatic) oxidation of the nucleotides. A 0.10-ml sample of the supernatant from this incubation was added to 0.15 ml of 10 N NaOH and allowed to incubate for 60 min at room temperature. To the remainder of the first incubation was added 0.05 U of NADase (Worthington Biochemicals Corp., Freehold, N.J.) in 1 M potassium phosphate buffer (pH 7.5), and the tube was reincubated for 2 h at 37°C. This procedure was experimentally determined to completely destroy 80 nmol of either NAD or NADP such that they would no longer form a fluorescent product. After treatment with NADase, a 0.10-ml sample of the solution was again treated with strong base. After incubation in 6 N NaOH, the samples (before and after NADase treatment) were diluted with 1.6 ml of water, and the fluorescence was measured in a Farrand A-4 fluorometer equipped with a 365-nm primary filter and a 448-nm secondary filter. The difference between the two fluorescence values was taken as the amount of NAD(P) formed in the first incubation. Standards were run in each experiment and subjected to the same treatment as all of the other samples. Activities are generally expressed as nanomoles of NAD(P)+ produced/30 min per 0.10 mg of protein. Other enzyme assays. The activities of two other granule-associated enzymes were determined as a function of substrate concentration. Assay conditions were exactly the same as those used for measurement of the oxidase activities, i.e., 0.1 M MES buffer (pH 6.0), 2.0 mM KCN, and 0.10 mg of granule fraction derived from resting or phagocytizing cells. Acid phosphatase activity was determined at varying levels of
t20 \
p-nitrophenylphosphate under these conditions. After a 30-min incubation, the reaction was stopped by the addition of 10 ml of 0.1 N NaOH, and the absorbance at 410 nm was measured on a Beckman DU spectrophotometer. A standard curve using known amounts of p-nitrophenol was used to calculate specific activities in terms of micromoles ofp-nitrophenol formed/30 min per 0.10 mg of protein. ,8-Glucuronidase activity was determined in a similar fashion, using varying concentrations of phenolphthalein glucuronide as substrate. Because of the very low activity of this enzyme under the incubation conditions, it was necessary to use a prolonged incubation time (20 h). At the end of this time the reaction was stopped by the addition of 3.0 ml of 0.34 M glycine-NaOH buffer (pH 10.0), and the absorbance was measured at 540 nm. A standard curve of known concentrations of phenolphthalein was used to calculate the specific activity as micromoles of phenolphthalein formed/20 h per 0.10 mg of protein.
RESULTS Figure 1 shows the effect of varying pH on the oxidation of NADPH determined by the isotopic assay procedure. This experiment was run with 0.17 mM NADPH in the presence of 2.0 mM cyanide. The normal phagocytizing granule fraction shows substantial activity above the spontaneous control activity, with a pH optimum very close to 6.0. A buffer effect is also noticed; MES shows greater activity than acetate, whereas phosphate demonstrates the gre:.test activity. In contrast, the phagocytizing granule fraction from a patient with CGD fails to show activity above the spontaneous oxidation over the entire pH range studied. It should be emphasized that the pH values listed were experimentally determined on the actual incubation mixture and are not simply the nominal pH of the buffer used. A comparison of the pH of the solutions at the beginning and end of the 30-min incubation showed that the pH did not change CGO GRAN Fx
NORM. GRAN Fx
CONTROL
l5
1,
