A continuous fluorescence assay for sulfhydryl oxidase

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 307 (2002) 266–272 www.academicpress.com

A continuous fluorescence assay for sulfhydryl oxidase Sonali Raje, Nicole M. Glynn, and Colin Thorpe* Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA Received 23 January 2002

Abstract Flavin-dependent sulfhydryl oxidases represent a newly discovered family of proteins with a range of cellular locations and putative roles. The avian and mammalian proteins can catalyze the direct oxidation of protein cysteine residues to disulfides with the reduction of dioxygen to hydrogen peroxide. Although thiols interfere with the peroxidase-mediated quantitation of hydrogen peroxide, a very sensitive, continuous fluorescence assay of the sulfhydryl oxidases can be devised with careful selection of thiol substrate concentration and fluorogen. Purified avian enzyme (or crude chicken egg white) was used for these experiments. Homovanillic acid was found to be a suitable fluorogen in the presence of 300 lM thiols from either dithiothreitol or reduced ribonuclease A. High concentrations of horseradish peroxidase minimized the effects of contaminating catalase in biological samples. Using fluorescence microcells, the assay could detect 15 fmol of avian sulfhydryl oxidase and the rates were linearly dependent on enzyme concentration up to 6 nM. Aspects of the interaction among thiols, homovanillic acid, and peroxidase are discussed which limit the sensitivity of the assay and require that care is exercised in the application of this new procedure. Finally, the assay is used to show that there is sulfhydryl oxidase activity in a number of secretory fluids including human tears. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sulfhydryl oxidase; Fluorescence assay; Protein disulfide bond formation

There has been a recent resurgence of interest in enzymes that catalyze the formation of disulfide bridges at the expense of molecular oxygen: 2P–SH þ O2 ! P–S–S–P þ H2 O2 : Two categories of sulfhydryl oxidases have been described. Both metal- [1–3] and FAD-dependent [4–10] classes have been shown to catalyze the direct oxidation of protein thiols without the mediation of small molecules such as glutathione. Sequencing of avian [9] and murine [11] flavin-linked sulfhydryl oxidases shows that they represent founding members of a family of enzymes that are found in all multicellular genomes sequenced to date. A human analog, Quiescin Q6, has sulfhydryl oxidase activity [9] and is secreted from fibroblasts as they enter reversible quiescence [12–14]. The sequence of humanbone-derived growth factor and cell inhibitory factor indicates that they are also apparently sulfhydryl oxidases [9,11]. A domain of these flavoenzymes, containing the

*

Corresponding author. Fax: 302-731-6335. E-mail address: [email protected] (C. Thorpe).

redox-active disulfide, has been recently found to have stand-alone sulfhydryl oxidase activity in yeast (as proteins essential for respiration and vegetative growth: ERV11 and ERV2 [15–18]) and mammals (as augmenter of liver regeneration, ALR [19]). Further, these smaller flavoenzymes have a homolog in the genome of poxviruses that participates in the introduction of disulfide bridges in the viral envelope [20,21]. The development of a highly sensitive, continuous assay would be very helpful in exploring this emerging gene family. A widely used assay for the sulfhydryl oxidases involves monitoring the disappearance of thiols using discontinuous sampling with DTNB [22]. This is both simple and versatile, but it does entail following small differences in a large background absorbance (particularly when Km values for some thiol substrates are >5 mM [2,4–6]). Quantitation of thiols via fluorescent 1

Abbreviations used: DTNB, 5,50 -dithio-bis(2-nitrobenzoic acid); DTT, dithiothreitol; GSH, reduced glutathione; HVA, homovanillic acid; ERV, essential for respiration and vegetative growth; ALR, augmenter of liver regeneration; EDTA, ethylenediaminetetraacetic acid; ABTS, 2, 20 -azino-bis1 (3-ethylbenzothiazine-6-sulfonic acid).

0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 2 6 9 7 ( 0 2 ) 0 0 0 5 0 - 7

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reagents would improve the sensitivity, but not address the issue of the large starting signals from which small decreases are to be discerned. Mixing capillary streams of thiol-containing reaction mixture and DTNB in a flowcell could yield a continuous absorbance assay but again does not circumvent the issue of sensitivity and background absorbance. Finally, monitoring oxygen consumption polarographically is convenient and widely employed for the sulfhydryl oxidases [4,6,23,24], but again the disappearance of a substrate is followed via a method that is intrinsically not very sensitive. This paper addresses the continuous monitoring of hydrogen peroxide generated by sulfhydryl oxidase using horseradish-peroxidase-mediated generation of a fluorogen. While this may seem an obvious approach, many thiol compounds markedly or completely suppress signal generation from both chromophoric or fluorogenic reagents via the interception of radical intermediates or products of the reaction [25,26]. Thus, Sliwkowski and Swaisgood [23], citing earlier work of Randall on benzidine [27], showed the impracticability of using 2,20 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to follow hydrogen peroxide generation by the milk sulfhydryl oxidase. Here quenching of the radical signal appears almost quantitative [23]. Such interferences extend to modern, highly sensitive, fluorogenic reagents such as Amplex Red, whose use is compromised2 by concentrations of thiols greater than 10 lM. One way to avoid this interference is to alkylate residual sulfhydryl groups before adding the chromogenic or fluorogenic reagent [23,28,29]. This procedure is necessarily discontinuous with the sulfhydryl oxidases and may lead to depletion of hydrogen peroxide prior to sampling via nonenzymatic or enzymatic processes. This paper shows that peroxidase-mediated dimerization of homovanillic acid (HVA) is less sensitive to thiol interference than the reagents listed above. HVA was introduced by Guilbault and co-workers [30] and reacts with peroxidase as shown in Fig. 1. This reagent was used in a coupled assay for acyl-CoA synthetase [31] in the presence of 100 lM CoASH and so this suggested that a workable continuous fluorescence method for the sulfhydryl oxidases might be devised. Here we show that an HVA-based assay can measure 100 pM concentrations of avian sulfhydryl oxidase (15 fmol of enzyme in fluorescence microcells). This sensitivity should find application with a range of sulfhydryl oxidases including the recently described ERV1, ERV2, and ALR gene products [15–19]. Although the assay is practical, rapid, and convenient, it must be used with attention to certain complexities in the interaction between components in the assay.

2 Amplex Red (Molecular Probes) hydrogen peroxide assay kit (A-12212) product information sheet.

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Fig. 1. Peroxidase-catalyzed oxidation of homovanillic acid by hydrogen peroxide.

Materials and methods DTT, DTNB, GSH, HVA, RNase A, catalase, D glucose, and glucose oxidase were purchased from Sigma. Horseradish peroxidase was purchased from Boehringer Mannheim. Hydrogen peroxide was obtained as a 30% solution from Fisher Scientific. Amplex Red was purchased from Molecular Probes. Purified sulfhydryl oxidase was a generous gift from Dr. Karen Hoober. PD-10 desalting columns were obtained from Amersham Pharmacia Biotech. Animal tissues were purchased from Pelfreeze Biologicals. GSH and DTT solutions were prepared daily in water and standardized with DTNB using a molar extinction coefficient of 13.6 mM1 cm1 for the thiolate anion at 412 nm [22]. When concentrated solutions of GSH were included in assays, stocks were made in 50 mM phosphate buffer and brought to pH 7.5 with KOH. RNase (25 mg) was reduced using a twofold molar excess DTT over total protein thiols in 1 mL of 6.0 M guanidine hydrochloride prepared in deoxygenated phosphate buffer and adjusted to pH 7.5. The mixture was incubated under nitrogen at 37 °C for 5 h prior to desalting using a PD-10 column (Sephadex G25 M; Amersham Pharmacia Biotech) equilibrated with 0.1% acetic acid. The elution was monitored to ensure complete separation of reduced RNase from excess DTT. The thiol titer was determined using DTNB and a molar extinction coefficient of 9400 M1 cm1 at 280 nm for reduced RNase [32]. Pooled RNase (1–3 mM protein thiols in 0.1% acetic acid) was distributed in several small glass tubes placed within a larger container. The vessel was subject to multiple cycles of evacuation and flushing with nitrogen before being stored under nitrogen [33]. Samples of reduced RNase were withdrawn as needed. The decline in thiol titer of the stored material was insignificant over many weeks at 4 °C. Hydrogen peroxide was freshly diluted and standardized daily using a molar extinction coefficient of 43.6 M1 cm1 at 240 nm [34]. Horseradish peroxidase, glucose oxidase, and sulfhydryl oxidase were quantitated using molar extinction coefficients of 108 mM1 cm1 at 404 nm [35], 14.1 mM1 cm1 at 454 nm [36], and 12.5 mM1 cm1 at 454 nm [6], respectively. Biological tissues (1 g each) were triple rinsed in cold phosphate-buffered saline and finely diced on an ice-cold platform. They were then mixed with 10 mL of phosphate buffered saline and homogenized with a Tissue

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Tearor from Biospec Products at 30,000 rpm for 5 min on ice. After centrifugation at 14,000 rpm in a benchtop Eppendorf centrifuge, the supernatants were collected and assayed immediately or stored frozen at )20 °C. The egg white from one chicken egg was diluted with 2 vol of 50 mM phosphate buffer and thoroughly homogenized to break up the thin and thick layers [6]. After centrifugation to remove suspended material [6], the supernatant was used immediately or distributed in plastic containers for storage at )20 °C. All assays were performed at 25 °C in 50 mM potassium phosphate buffer at pH 7.5 containing 0.3 mM EDTA unless otherwise stated. The final assay mixture contained 1 mM HVA, 1.4 lM horseradish peroxidase, and 300 lM thiols from either DTT or RNase. In the fluorescence microcell assay utilized here, 93 lL from a premixed cocktail of buffer, HVA, and peroxidase, kept on ice, was added to the cuvette (Starna Cells, Atascadero, CA) and allowed to warm to 25 °C. Thiol and sufficient water were added to bring the complete reaction mixture (anticipating enzyme addition) to 150 lL and the baseline was recorded for about 1 min (see later). Note that the thiol substrate was omitted from the stock cocktail to minimize a very slow peroxidase-mediated thiol oxidation [37] prior to the addition of sulfhydryl oxidase. This reaction with DTT [37] and, to a lesser extent reduced RNase, results in a progressive increase in the starting fluorescence for any given series of assays. Assays were started by the addition of 1–10 lL of enzyme to the cuvette and thorough mixing of the contents using a clean micropipette tip. The assay was checked at the beginning and end of each series of assays by the addition of 1–5 lM hydrogen peroxide instead of sulfhydryl oxidase. The resulting step in fluorescence was a measure of reproducibility and was one measure of the effectiveness of thiols to depress the appearance of fluorescent product (see later). Fluorescence assays were performed in an SLMAminco Bowman Series 2 luminescence spectrometer. The sample holder and the reaction cocktail were maintained at 25 °C using a circulating waterbath. The instrument photomultiplier voltage was set using phosphate buffer in fluorescent microcells using the parameters kex 350 nm (1-nm slit width) and kem 397 nm (8-nm slit width) at a sensitivity of 3% of the full scale. Thereafter, the increase in fluorescence for the products of the HVA and Amplex Red reagents was followed at kex 320 nm, kem 420 nm and kex 560 nm, kem 590 nm, respectively. The assay can be readily adapted to semimicrocells by increasing the volume of reagents used. Visible and ultraviolet spectra were recorded on a Hewlett–Packard 8452A diode array spectrophotometer. Oxygen electrode assays were performed as before [6] in a Clark-type oxygen electrode (YSI 5331) using 5 mM DTT in 3 mL of air-saturated phosphate buffer, pH 7.5.

Results and discussion In preliminary experiments we confirmed the observations of Sliwkowski and Swaisgood [23] that assays of sulfhydryl oxidase using glutathione and the peroxidasemediated generation of the radical cation of ABTS are unworkable. Thus, when 20 lM hydrogen peroxide was added to 100 lM ABTS (in 50 mM phosphate buffer, pH 7.5, containing 0.6 lM peroxidase) in the presence or absence of 300 lM of DTT or RNase thiols 0 or 2% of the expected absorbance appearance at 640 nm was observed (not shown). The highly sensitive and generally versatile fluorogenic reagent Amplex Red is reported to be compromised by thiol concentrations greater than 10 lM. Guilbault and co-workers have used a series of aromatic acids as fluorogenic reagents for peroxidase mediated assays [30,38] and we have chosen HVA here for the bulk of our experiments. However, preliminary

Fig. 2. Continuous fluorescence microcell assay of sulfhydryl oxidase using reduced RNase as substrate. A shows assay traces before and after the addition of 2.6 nM pure avian sulfhydryl oxidase (trace 1) or 6 lL of 1:3 diluted egg white (trace 2) to the assay mixture containing 300 lM RNase thiols (see Materials and Methods). Initial rates as a function of the concentration of pure sulfhydryl oxidase are shown in B. The inset extends the concentration range down to 0.1 nM.

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studies show that p-hydroxyphenylacetic acid [38] would also be suitable for the work developed here. Fig. 2A represents two sulfhydryl oxidase assays following the appearance of the fluorescent HVA dimer in microcells. We use microcells here (150-lL assay volume) both to maximize the sensitivity and to minimize the use of reagents, although semimicro or full-size 1-mL cuvettes could be employed if desired (see Materials and Methods; not shown). RNase (300 lM thiols; see Materials and Methods) is the oxidizable substrate in Fig. 2. The low background rate was achieved by decreasing the intensity of the exciting incident light, thereby minimizing a slight light-mediated increase in fluorescence in the assay mixture minus oxidase. Sensitivity was maintained by a compensatory widening of the exit slits (see Materials and Methods). Trace 1 in Fig. 2A represents the addition of 2.6 nM purified avian sulfhydryl oxidase and shows a satisfactorily linear increase in fluorescence at 420 nm due to the appearance of the HVA dimer [30]. Relative rates are plotted as a function of the concentration of the purified enzyme in panel B down to 100 pM sulfhydryl oxidase. This lower limit corresponds to 15 fmol of purified oxidase in the cuvette, an amount some 1000-fold smaller than that required to obtain detectable rates in the 3-mL assay volume of a standard oxygen electrode device. Since purified sulfhydryl oxidase is not yet commercially available, trace 2 in Fig. 2A was obtained with 6 lL of a three-fold dilution of chicken egg white. The viscosity of untreated egg white makes dilution and homogenization of the sample desirable before it can be reproducibly delivered into fluorescence microcells (see Materials and Methods). Thus, normal egg white provides a convenient and relative abundant source of the enzyme to allow validation of the assay in the absence of pure stocks of sulfhydryl oxidase. The application of the assay to crude tissue samples is described later. Oxygen electrode assays showed that DTT is a good substrate of both the avian and the mammalian flavinlinked sulfhydryl oxidases [4,6]. Fig. 3A shows the corresponding HVA fluorescence assay of the purified avian enzyme using 150 lM DTT (300 lM thiols). Again, these DTT assays readily allow initial rates to be determined, and these rates are plotted with enzyme concentration in Fig. 3B. Thus, the HVA fluorescence assay, under the conditions described here, is convenient and applicable to two diverse substrates of the oxidase. However, attempts to use glutathione with the avian oxidase show that the assay is not suitable for substrates with relatively high Km values for a particular sulfhydryl oxidase. For example, glutathione (Km ¼ 20 mM; [6]) must be present at multimillimolar concentrations in assays of the avian enzyme, but these levels result in an unacceptable increase in the background rate before the addition of oxidase (not shown). In this case, a direct peroxidase-mediated oxidation of thiol groups occurs as reported earlier [37]. Similarly, b-mercaptoethanol

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Fig. 3. Fluorescence assays of sulfhydryl oxidase using DTT as substrate. A shows an assay trace using 1.3 nM oxidase using 150 lM DTT (300 lM thiols; see Materials and Methods). Initial rates are plotted as a function of pure sulfhydryl oxidase concentration in B.

cannot be used with the avian enzyme (Km ¼ 54 mM [6]). In contrast, this background rate is minimized at the low concentration (300 lM thiol) of RNase and DTT used in Figs. 2 and 3 allowing low oxidase-mediated rates to be reliably discerned. In addition to effects on the background rate before the addition of sulfhydryl oxidase, increasing thiol concentrations lower the yield of fluorophore by competing for the radical intermediate(s) of the peroxidase reaction. Fig. 4 presents a simplified scheme of this rather complex behavior. HRP in the presence of hydrogen peroxide converts two HVA molecules to radicals (X; step 2) [30,39]. A second-order dimerization of radicals generates the desired fluorescent dimer (X-X; step 3) in competition with a first-order interception of the radicals by RSH (step 4; see later [40–43]). One illustration of the interference of thiols with HVA dimer appearance is presented in Fig. 5. Here, hydrogen peroxide is generated using glucose oxidase/ glucose in an otherwise identical fluorescent assay system that was used for sulfhydryl oxidase. d-glucose þ O2 ! d-glucono-d-lactone þ H2 O2 As before, the liberated hydrogen peroxide is coupled to HVA dimer production using peroxidase and the

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Fig. 4. Simplified scheme showing interference by thiols in the peroxidase-mediated formation of HVA dimers (see the text).

Fig. 5. Effect of DTT and RNase on the apparent activity of glucose oxidase followed by oxygen electrode and HVA dimer formation. A shows the effect of increasing DTT thiol concentration on the initial assay rates in the oxygen electrode (d); 3.9 nM glucose oxidase, 20 mM D -glucose in 3 mL of 50 mM phosphate buffer, pH 7.5) and in the HVA assay (r; using 20 mM D -glucose with 0.5 nM glucose oxidase in 0.15 mL phosphate buffer). In B, RNase replaces DTT thiols under otherwise identical conditions.

percentages of control activity are plotted as a function of DTT concentration. Increasing DTT concentration exerts a biphasic apparent inhibition in rate with 55 and 15% signal remaining at 100 lM and 10 mM thiols, respectively (Fig. 5A). There is no comparable DTT-mediated loss of activity when glucose oxidase is assayed in the oxygen electrode (Fig. 5A). The actual slight increase in activity reflects contamination of commercial glucose oxidase with small levels of sulfhydryl oxidase (not shown) as reported earlier [5]. Fig. 5B shows that reduced RNase also exerts noticeable impact on the rates of HVA dimer formation with glucose oxidase amounting to 20 and 45% reduction at 100 and 300 lM protein thiols, respectively. Clearly, while the new assay can conveniently quantitate enzyme (Figs. 2 and 3), the present procedure cannot be used for the determination of the Km values of thiol substrates. In choosing a suitable concentration of thiol substrate to use for routine quantitation of sulfhydryl oxidase using this new fluorescent assay, one must balance the benefits of using saturating substrate with the decreased sensitivity accompanying increased thiol concentrations. For the avian enzyme, the concentration of 300 lM thiols for DTT (at the Km ) and reduced RNase (at twice the Km ) is a suitable compromise affording satisfactory linearity and adequate rates with these varied substrates. The quenching of HVA radicals (Fig. 4, step 4) is terminated by a complex radical chain reaction (step 5) that leads to consumption of thiol and oxygen and the generation of disulfide and reduced oxygen species including hydrogen peroxide and superoxide anion [44– 49]. This reaction leads to the depletion of substrate thiol in a sulfhydryl-oxidase-independent manner. This side reaction likely contributes to the slight curvature of the assay trace in Fig. 3A. Thus, when an assay was run over 10 min the increase in fluorescence corresponds to an apparent generation of 3 lM H2 O2 . However, DTNB assays of thiol titer reveal a corresponding 25 lM drop in DTT concentration (50 lM thiols in a total of 300 lM) over this time interval. At the outset we wanted to develop an assay that was relatively resistant to the levels of catalase that might be realistically present in crude extracts. This was achieved using a large concentration (1.4 lM) of horseradish peroxidase in the standard assay. Under these conditions the addition of a large concentration of catalase (880 nM final concentration in the assay; 290 units/mL) decreased the rate of HVA dimer formation by only 30% (Fig. 6, traces 1 and 2). This level of catalase is unlikely to be encountered in sulfhydryloxidase-containing extracts. The competition between peroxidase and 0.88 lM catalase is graphically illustrated by the almost complete disappearance of HVA dimer formation when the peroxidase concentration is reduced 10-fold (to 0.14 lM; Fig. 6: compare curves 3 and 4).

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Thus, providing that thiol substrates exhibiting low Km values for the sulfhydryl oxidase of interest are selected, the new continuous assay represents a convenient and sensitive method of enzyme quantitation for this emerging new family of enzymes and growth factors [9,11,15–17,19,51].

Acknowledgments We thank Professor Christine C. Winterbourn for her critical insight on radical scavenging reactions of thiol compounds and Professor Ian H. Mather for the milk-fat globule membrane supernatant. This work was supported by NIH Grant GM26643.

Fig. 6. Catalase interference in peroxidase-mediated sulfhydryl oxidase assay. Trace 1 represents a standard assay with 5 lL of diluted egg white (see Materials and Methods) added at 100 s. Trace 2 repeats the experiment after the addition of a large concentration of added catalase (0.88 lM; 290 units/mL). Traces 3 and 4 are the corresponding assays using a 10-fold lower peroxidase concentration (0.14 lM) showing almost complete suppression of HVA dimer formation upon the addition of catalase.

Table 1 Sulfhydryl oxidase activity in tissues and secretions using DTT and RNase Sample type

Chicken egg white Pig kidney Bovine seminal vesicle Milk-fat sup Human tears Laying hen magnum

Amount of H2 O2 produced: nmol min1 mg1 of protein Substrate: DTT

RNase

0:875  0:100 0:010  0:001 0:050  0:010 0:130  0:003 0:023  0:001 0:880  0:020

0:990  0:160 0:006  0:0 0:016  0:003 0:030  0:001 0:060  0:001 1:392  0:020

Table 1 shows the activity in several biological samples assayed using both DTT and reduced RNase as substrates. The specific activity of chicken egg white and the homogenates from the magnum of the oviduct from a laying hen is comparable. These samples show the highest specific activity among those studied to date. However, there is appreciable activity from a range of other sources including porcine kidney, bovine seminal vesicles, bovine milk-fat globule membrane supernatants [50], and even human tears. Milk contains an irondependent sulfhydryl oxidase [1] and so it appears that the assay described here can be used for the metalloenzyme as well as the flavin-linked proteins. While the purpose of this paper is not a comprehensive survey of sulfhydryl oxidase levels in tissues and secretory fluids, Table 1 supports the suggestion that sulfhydryl oxidase activity is widely distributed in nature [4,9,11,13,14].

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