A microtiter-plate assay of nitric oxide synthase activity - Springer Link

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John Dawson and Richard G. Knowles. Abstract. We describe here a microtiter-plate assay for measuring nitric oxide synthase (NOS) activity by utilizing.
Microtiter-Plate Assays

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A Microtiter-Plate Assay of Nitric Oxide Synthase Activity John Dawson and Richard G. Knowles

Abstract We describe here a microtiter-plate assay for measuring nitric oxide synthase (NOS) activity by utilizing the spectral shift in optical absorbence between the wavelengths 405 and 420 nm on conversion of oxyhemoglobin to methemoglobin by nitric oxide (NO). This is a high-throughput assay permitting 96 or 384 simultaneous kinetic measurements and is ideal for the study of NOS inhibitors and their time dependence. It is also possible to measure enzyme rates under different conditions simultaneously for the study of the cofactor and substrate dependence of NOS preparations. The assay requires approximately l0 pmol/min of NOS activity to achieve a 1moD/min rate. Index Entries: Nitric oxide synthases; oxyhemoglobin; microtiter-plate; kinetic.

NOS activity and increase the sensitivity, and has now been further modified to permit it to be run in microtiter-plate format. The original assay (2) used the absorbence difference between 401 nm (maximum point) and 411 nm (isosbestic point); however, greater sensitivity is achieved by monitoring the absorption difference between the wavelengths 401 and 421 nm (minimum point). This microtiter-plate assay uses the closest readily available filters to do this, 405 nm and 420 nm (giving close to the maximal sensitivity), and uses a relatively full well (250 µl total volume) to maximize the optical path-length and thereby the sensitivity. With the appropriate microtiter-plate reader, this assay permits 96 or 384 simultaneous kinetic measurements of NOS activity. It is run at 37°C and is ideal for the study of NOS inhibitors and their time dependence, which can also be temperature-dependent. Using a similar method, it is possible to measure enzyme rates under different conditions simultaneously. This is useful, for

1. Introduction Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine, molecular oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH) to NO, citrulline, and NADP+ (reviewed in ref. 1). The neuronal (n) and endothelial (e) NOS isozymes are highly regulated by Ca2+ and calmodulin (CaM), whereas the iNOS has CaM tightly bound. NOS are heme proteins that also contain tightly bound flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and require tetrahydrobiopterin (BH4) for activity, whereas NADPH is used as a substrate. This article describes a microtiter-plate assay for measuring NOS activity by utilizing the spectral shift in optical absorbence on conversion of oxyhemoglobin to methemoglobin by nitric oxide (NO). The oxyhemoglobin assay was first described by Feelish and Noack to quantify NO release by chemical NO donors (2), was modified by Salter and Knowles (3) to apply it to measure

*Author to whom all correspondence and reprint requests should be addressed. Glaxo-Wellcome Research and Development, 5G120, Gunnels Wood Road, Stevenage, Herts SG1 2NY. E-mail jd43048@ Glaxowellcome.Co.UK Molecular Biotechnology ¹1999 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/1999/12:3/275–279/$11.25

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example, for the study of the cofactor and substrate dependence of NOS preparations. Other microtiter-plate assays of NOS activity have been described. The oxymyoglobin assay (4,5) should on theoretical grounds be very similar to that described here, kinetically measuring NO formation. Assays of NOS by measuring NADPH oxidation (6) are much less sensitive and may not accurately reflect NO synthesis, since NADPH consumption is under some circumstances uncoupled from NO synthesis. Assays of NOS using the Griess reaction to measure nitrite formed (7) are end-point rather than kinetic, are somewhat less sensitive, and are subject to the concern that only one of the two major products of NO breakdown (nitrate and nitrite) is being measured: the ratio of nitrite to nitrate formed can vary depending on what is present in the assay. Microtiter-plate assays based on oxyhemoglobin or oxymyoglobin are therefore the most sensitive and reliable assays for NOS activity. This version of the assay is somewhat less sensitive than the optimized spectrophotometric (3) or radiometric (8) assays, requiring approx 10 pmol/min of NOS activity to achieve a 1 mODU/min rate. This sensitivity is quite adequate for many studies, e.g., assays of brain-cytosol NOS, of recombinant-expressed NOS, or of purified NOS.

2. Materials 1. Extraction buffer (EB): The extraction buffer should be prepared in advance. The basic buffer is first prepared by dissolving sucrose (250 mM), Tris base (50 mM), and ethylenediaminetetraacetic acid (EDTA) (1 mM) in double-distilled or milliQ-grade water and bringing its pH to 7.4 at room temperature by the addition of HCl. The following constituents are then added to the final concentrations indicated: 0.1 mM D/Ldithiothreitol (DTT), 0.5 µM leupeptin, 0.5 µM pepstatin A, and 10 µM Antipain; the buffer is then made up to its final volume with water. This EB is then distributed into aliquots (typically 50 mL per aliquot) and stored at –20°C until required.

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2. 10 mg/mL Phenylmethylsulphonyl fluoride (PMSF): Because it is unstable in aqueous solution, PMSF is not included in the buffer at this stage, but prepared as a solution in absolute ethanol, stored at –20°C, and added to the EB during the extraction procedure (see below). The composition of this EB is designed to permit extraction of NOS from cells or tissues without breaking intracellular organelles and minimizing proteolysis by chelating divalent cations with EDTA and the inclusion of protease inhibitors. 3. Assay buffer: 100 mM HEPES is dissolved in double-distilled or milliQ-grade water and brought to pH 7.4 by the addition of NaOH. This can be stored for several weeks at 4°C. Prior to the assay of NOS, DTT is added to the buffer to give 100 µM. CaCl2, MgCl2, and hemoglobin are added as required and the buffer mixture warmed to the required temperature. The cofactor and substrate stocks are kept in the dark on ice until use (see Notes 1–3). 4. 1 M stock solutions of CaCl2 and MgCl2 can be stored for several weeks at 4°C. 5. Cofactor stocks: 7.5 mM L-arginine (HCl), 250 mM NADPH, 250 µM FMN, 250 µM FAD, and 25 µM CaM (Sigma P2277, from Bovine Brain, assumed M.W. 17,000) are dissolved in HEPES buffer and stock solutions kept at -20°C. A “cocktail” of these can be made and stored in the same way to reduce the number of additions to the assay. 6. (6R)-5,6,7,8-Tetrahydro-L -biopterin hydrochloride (BH4): On the day of assay, DTT is added to 10 mM HCl (stock stored at room temperature) to give 500 µM. BH4 is dissolved in this to give 500 µM and the solution kept on ice in the dark until use. It can be used for approx 4 h. 7. NOS inhibitors: Inhibitors are dissolved in 25% (v/v) DMSO/100 mM HEPES buffer pH 7.4 at 12.5 times the final concentration required. A stock solution of 12.5 mM NG-methyl-L -arginine ( L -NMMA) for blanks, dissolved in this way, can be stored at –20°C. Up to 2% DMSO can be tolerated in the assay. For IC50 determination, we routinely use 10 concentrations with twofold dilution. Volume 12, 1999

Microtiter-Plate Assays 8. Ion-exchange resin: Dowex 50W Na + form, 200–400 mesh, 8% cross-linked. This is prepared by converting the H + form (Sigma 50X8–400) by washing the resin in a beaker with 1M NaOH. Two washes with 1 vol of NaOH are carried out by adding NaOH to the resin, mixing several times, allowing the resin to settle under gravity (~5 min) and aspirating the supernatant with a water-jet vacuum pump. The resin is then washed with water until the pH is