Cell Biology Biochemistry

Chemistry Protein Structure & Function

Pharmacology General Methods

Biochemistry

Enzymes: Absorbance

• Adenylate Kinase/NADH
• Alcohol Dehydrogenase, Glutathione S-transferase & Esterase Activity
• β-galactosidase
• β-lactamase
• Caspase-3
• Catalase & Glutathione S-transferase
• Elastase
• Glutathione, Myeloperoxidase & Malondialdehyde Assays
• Lactate Dehydrogenase
• NADPH Oidase/Cytochrome C Reduction
• P450 Enzymes

Enzymes: Fluorescence

• Caspase-3

adenylate kinase/NADH

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

J. Cell. Biol. 147: 809–22 (1999)

Ruth M. Kluck¹, Mauro Degli Espostie, Guy Perkins², Christian Renken³, Tomomi Kuwana¹, Ella Bossy-Wetzel¹, Martin Goldberg⁴, Terry Allen⁴, Michael J. Barber⁵, Douglas R. Green¹, and Donald D. Newmeyer¹.
¹ Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121.
² Department of Neurosciences, University of California San Diego, San Diego, California 92093
³ Biology Department, San Diego State University, San Diego, California 92182.
⁴ Paterson Institute, Christie Hospital NHS Trust, Manchester M20 9BX, United Kingdom.
⁵ Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, Florida 33612.

Methods: mitochondrial AK activity assay. Mitochondrial AK activity was measured by a modification of the method of Schmidt et al. 1984. Extract aliquots (50 µL) containing ~0.05 mg mitochondrial protein were pelleted at 12,000 g for 3 minutes, and the pellet washed twice in 800 µL buffer D (60 mM sucrose, 210 mM mannitol, 10 mM KCl, 0.5 mM DTT, 10 mM succinate, 10 mM HEPES/KOH, pH 7.5, and 5 mM EGTA) to remove contaminating cytosolic AKs. The mitochondrial pellets were lysed with 50 µL of 1% Triton X-100 in buffer D to release remaining AK, and the sample stored at –80 °C. AK activity was measured in a mixture composed of 1 mL of 130 mM KCl, 6 mM MgSO4, 100 mM Tris-HCl pH 7.5, 15 µL 0.1 M NADH, and 5 µL each of 0.1 M ATP, 100 mM phosphoenol pyruvate, 1 mM rotenone, 1.5 mM oligomycin, a mixture of pyruvate kinase and lactate dehydrogenase (80 U/mL each), and 0.15 M AMP. 200 µL of buffer mix was added to 6 µL of sample in a microtiter plate. The absorbance decrease of NADH was measured at 366 nm in a microtiter plate reader (SpectraMax 250, Molecular Devices Corp.) for 10 minutes at 22 °C. The rates were calculated (SoftMax Pro) and calibrated against chicken muscle myokinase (Sigma) activity. Rates obtained in the presence of the inhibitor di-adenosine pentaphosphate (400 µM; Sigma) were minimal and subtracted as background.

Quantitative kinetic assays for glutathione S-transferase and general esterase in individual mosquitoes using an EIA reader

Insect Biochem. 19:741–751 (1989).

David F. Grant, Daniel M. Bender, and Bruce D. Hammock.
Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616 USA.

Summary. Microassays for alcohol dehydrogenase (ADH), glutathione S-transferase (GST), general esterase (EST), and total protein have been applied to the analysis of insecticide resistance in individual mosquitoes. The microassays were performed in a VMax kinetic microplate reader and the results were compared to previously established spectrophotometer based protocols. The microassays run in parallel increased the efficiency of screening large numbers of samples. The advantages of running multiple enzyme assays on individual insects is discussed in light of theories on the molecular biology of insecticide resistance.

Action of the thiamine antagonist bacimethrin on thiamine biosynthesis

J. Bacteriol. 182: 5606–10 (2000).

Julie L. Zilles, Laura R. Croal, and Diana M. Downs.
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706.

Methods: β-galactosidase activity. Subcultures were grown to 0.3 to 0.4 A650 before being assayed. β-Galactosidase activity was assayed as previously described, except that volumes were reduced 10-fold, and absorbance was read in a microtiter plate using a SpectraMax Plus microplate reader (Molecular Devices Corp.)

HveA (herpesvirus entry mediator A), a coreceptor for herpes simplex virus entry, also participates in virus-induced cell fusion

J. Virol. 72: 5802–10 (1998).

Tracy Terry-Allison¹, Rebecca I. Montgomery¹, J. Charles Whitbeck^2,3,4, Ruliang Xu^2,3,4, Gary H. Cohen^2,3, Roselyn J. Eisenberg^3,4, and Patricia G. Spear¹.
¹ Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois ⁶⁰⁶¹¹.
² School of Dental Medicine, Northwestern University Medical School, Chicago, Illinois 60611.
³ Center for Oral Health Research, Northwestern University Medical School, Chicago, Illinois 60611.
⁴ School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.

Methods: β-galactosidase activity. Cells were reseeded into both 96-well culture dishes (quantitative analysis) and 24-well culture dishes (qualitative analysis) in the absence or presence of various concentrations of anti-HveA serum or pre-immune control serum control. Approximately 24 hours after plating, the 24-well plates were fixed, permeabilized, and overlaid with ferricyanide buffer supplemented with X-Gal (0.5 mg/mL; Gibco BRL), while the 96-well plates were rinsed with PBS and solubilized in PBS-0.5% NP-40 supplemented with o-nitrophenyl-b-D-galactopyranoside (ONPG; Sigma) at 3 mg/mL. Plates were read in a SpectraMax 250 (Molecular Devices Corp.)

A microtiter-based assay for the determination of ID50s of β-lactamase inhibitors employing reporter substrates detected at UV or visible wavelengths

Molecular Devices MaxLine Application Note #20.

David J. Payne and Sarbendra Pradhananaga
Department of Molecular Microbiology, SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Road, P.O. Box 5089, Collegeville, PA 19426-0989 USA

Methods: β-lactamase assays. β-lactamases are either plasmid or chromosomally encoded bacterial enzymes which hydrolyze β-lactam antibiotics. This application note describes a simple procedure to determine the inhibition (ID₅₀ value) of β-lactamases by various agents using the chromogenic cephalosporin nitrocefin (lmax = 482 nm) and the carbapenem antibiotic imipenem (lmax = 299 nm) as reporter substrates. Imipenem was used for those carbapenemases which did not have significant hydrolytic activity against nitrocefin. The method described uses the UV capability of the SpectraMax 250 microplate spectrophotometer and can be readily modified to determine ID₅₀ values of inhibitors of other enzymes requiring UV/Vis monitoring.

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

J. Cell Biol. 147: 809–22 (1999).
Ruth M. Kluck et al.

Methods: measurement of DEVDase activity. To measure caspase-3-like activation, extract aliquots (2 µL) were incubated with DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide, 40 µM; Biomol) in 200 µL of a buffer (250 mM sucrose, 20 mM HEPES/KOH pH 7.5, 50 mM KCl, 2.5 mM MgCl₂, and 1 mM DTT) similar to that used to make the egg extracts. Incubations were kept at 22 °C and A405 development monitored over 30 minutes (SpectraMax 250 microplate spectrophotometer, Molecular Devices Corp.)

Clusterin has chaperone-like activity similar to that of small heat shock proteins

J. Biol. Chem. 274: 6875–6881 (1999).

David T. Humphreys¹, John A. Carver², Simon B. Easterbrook-Smith³, and Mark R. Wilson¹.
¹ Department of Biological Sciences, The University of Wollongong, Northfields Avenue, Wollongong, New South Wales 2522, Australia.
² Department of Chemistry, The University of Wollongong, Northfields Avenue, Wollongong, New South Wales 2522, Australia.
³ Department of Biochemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.

Summary. Clusterin is a highly conserved protein which is expressed at increased levels by many cell types in response to a broad variety of stress conditions. At physiological concentrations, clusterin potently protected glutathione S-transferase (GST) and catalase from heat-induced precipitation and a-lactalbumin and bovine serum albumin from precipitation induced by reduction with dithiothreitol.

Methods: enzyme assays. Catalase was incubated with 1.0 mL of H₂O₂ substrate solution (0.12% (v/v) H₂O₂ in 50 mM Na₂HPO₄, pH 7.0) at 37 °C for 5 minutes before the reaction was stopped with 150 µL of 4 M NaOH. The absorbance of H₂O₂ was measured at 250 nm on a SpectraMax 250 microplate reader (Molecular Devices Corp.) Enzyme activity was measured as a decrease in absorbance. GST was heated at 37 or 50 °C for 30 minutes. GST was then diluted into substrate solution (1 mM GSH, 2 mM 1-chloro-2,4-dinitrobenzene in 0.1 M phosphate, pH 7.4) to a final concentration of 4.5 µg/mL and incubated at 37 °C for 5 minutes before measuring the absorbance at 350 nm. Enzyme activity was measured as an increase in absorbance, corresponding to the appearance of 1-S-glutathionyl-2,4-dinitrobenzene.

Inhibitors directed to binding domains in neutrophil elastase

Biochemistry 29: 9970–77 (1990).

Suresh C. Tyagi and Sanford R. Simon.
Department of Biochemistry and Cell Biology and Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York 11794.

Methods: human neutrophil elastase (HNE) activity. The amidolytic and HNE were assayed with MeOSucAAPVpNa as a substrate (Nakajima et al 1979). The release of p-nitroaniline was monitored by recording the absorbance at 405 nm with a VMax microplate reader (Molecular Devices Corp.) operating in the kinetic mode. The buffer used contained 3.3% dimethylformamide and 0.01% Triton X-100. Kinetic data was collected for five minutes, during which reaction rates remained linear.

A microassay method for neurotoxic esterase determinations

Fundam. Appl. Toxicol. 16: 110–116 (1991).

Linda Correll, and Marion Ehrich.
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061 USA.

Summary. The authors report new microassay methods for measuring the activities of neurotoxic esterase, EC 3.1.1 (NTE, also known as neuropathy target esterase) and acetylcholinesterase EC 3.1.1.7 (AChE) and total protein determinations in spinal cord regions of hens. NTE activity is reported as the change in phenol released from phenyl valerate as measured at 510 nm following the addition of 4-aminoantipyrine and potassium ferricyanide. Hydrolysis of acetylthiocholine iodide by AChE was followed as the change in absorbance at 412 nm as dithionitrobenzoic acid (DTNB) was reduced by the enzyme product.

The Bradford method read at 595 nm was used to measure total protein. Microassay methods were validated by comparison of NTE and AChE activity in brains of control and experimental hens with more conventional assays. NTE activities in brains of control and experimental hens given DFP (0.5 mg/kg sc) 24 hours before samples were collected produced similar results using both types of NTE analytical procedures. The reported advantages of the microassay include the capability of being used for small regional esterase activity measurements, the number of samples processed was increased while the time needed was reduced, and the total assay volume (0.3 mL) is 1/20 of sample and reagents necessary in more conventional assays.

Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples

Anal. Biochem. 190: 360–365 (1990).

Margaret A. Baker, George J. Cerniglia, and Aziza Zaman.
Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA 19104 USA.

Summary. The authors describe a rapid, sensitive, and easy assay for measuring reduced and oxidized glutathione (GSH and GSSG), both intracellular and extracellular in biological samples. Cell and tissue sample preparation for the GSH enzymatic recycling assay is described. The method follows 5,5´-dithio-bis(2-nitrobenzoic acid) (DTNB) reduction at 405 nm in the UVMax microplate reader. The kinetic microplate assay is sensitive to 5 pmol, uses less sample than previous methods, and enables increased sample processing.

Protection against hemorrhagic shock in mice genetically deficient in poly(ADP-ribose) polymerase

Proc. Natl. Acad. Sci. USA 97: 10203-10208 (2000).

Lucas Liaudet¹, Francisco Garcia Soriano³, Éva Szabó², László Virág², Jon G. Mabley², Andrew L. Salzman^1,2, and Csaba Szabó².
¹ Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229.
² Inotek Corporation, Suite 419 E, 100 Cummings Center, Beverly, MA 01915.
³ Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 01703.

Methods: myeloperoxidase (MPO) assay. Tissues were homogenized (50 mg/mL) in 0.5% hexadecyltrimethylammonium bromide in 10 mM 3-(N-morpholino)propanesulfonic acid (Mops) and centrifuged at 15,000 g for 40 minutes. The suspension was then sonicated three times for 30 seconds. An aliquot of supernatant was mixed with a solution of 1.6 mM tetramethylbenzidine and 1 mM hydrogen peroxide. Activity was measured spectrophotometrically as the change in absorbance at 650 nm at 37 °C by using a SpectraMax absorbance microplate reader (Molecular Devices Corp). Results are expressed as milliunits MPO activity per milligram of protein, which were determined with the Bio-Rad assay.

Methods: malondialdehyde (MDA) assay. MDA formation was used to quantify the lipid peroxidation in tissues and measured as thiobarbituric acid-reactive material. Tissues were homogenized (100 mg/mL) in 1.15% KCl buffer. Homogenates (200 µL) were then added to a reaction mixture consisting of 1.5 mL of 0.8% thiobarbituric acid, 200 µL of 8.1% (vol/vol) SDS, 1.5 mL of 20% (vol/vol) acetic acid (pH 3.5), and 600 µL of distilled H₂O and heated at 90 °C for 45 minutes. After cooling to room temperature, the samples were cleared by centrifugation (10,000 g for 10 minutes), and their absorbance was measured at 532 nm by using 1,1,3,3-tetramethoxypropane as an external standard. The level of lipid peroxides was expressed as nanomoles MDA per milligram of protein.

Methods: glutathione (GSH) assay. Tissues were homogenized (100 mg/mL) in 5% (vol/vol) sulfosalicylic acid. The homogenates were centrifuged at 10,000 g for 20 minutes, and an aliquot of the clear supernatant (20 µL) was combined with 0.3 M Na₂HPO₄ (160 µL) and 0.04% 5,5´-dithiobis-(2-nitrobenzoic acid) in 1% sodium citrate (20 µL). After a 10-minute incubation at room temperature, absorbance was read at 405 nm in a SpectraMax absorbance microplate reader. Concentrations of GSH were calculated from a standard curve constructed with known concentrations of reduced GSH.

Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica Serovar Typhimurium: implications for thiamine synthesis

J. Bacteriol. 182: 3896–3903 (2000).

Elizabeth Skovran and Diana M. Downs.
Department of Bacteriology, University of Wisconsin Madison, Madison, Wisconsin 53706.

Methods: lactate dehydrogenase assays. The lactate dehydrogenase assay was modified from the method of Oeschger. The reaction mixture contained pyruvate-specific D-lactic acid dehydrogenase (catalog no. L3888; Sigma Chemical Co.) (8.7 U), 10 mM Tris-HCl (pH 8), 240 mM KCl, 20 mM MgCl₂, and 1 mM pyruvate or 10 to 100 µL of spent growth medium. The preassay mixture was incubated at 30 °C for 2 minutes before the reaction was initiated with NADH (final concentration, 25 mM). The oxidation of NADH was monitored as a decrease in the absorbance at 340 nM in a quartz microtiter plate using a SpectraMax Plus microplate reader (Molecular Devices Corp.)

Mapping of functional domains in p47phox involved in the activation of NADPH oxidase by “peptide walking”

J. Biol. Chem. 273: 15435–44 (1998).

Igor Morozov, Ofra Lotan, Gili Joseph, Yara Gorzalczany, and Edgar Pick.
The Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

Methods: cell-free NADPH oxidase assay. The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47phox, p67phox, p40phox, and the small GTPase, Rac(1 or 2). O₂ production was initiated by the addition of NADPH and quantified by following the rate of cytochrome c reduction, at 550 nm, in a kinetic assay performed in a SpectraMax 340 microplate reader (Molecular Devices Corp.), using SoftMax Pro software.

Evidence for peroxisome proliferator-activated receptor (PPAR)-independent peroxisome proliferation: effects of PPAR-specific agonists in PPAR-null mice

Mol. Pharmacol. 58: 470-76 (2000).

John G. DeLuca², Thomas W. Doebber¹, Linda J. Kelly¹, Ramon K. Kemp², Sylvain Molon-Noblot2, Soumya P. Sahoo¹, John Ventre¹, Margaret S. Wu¹, Jeffrey M. Peters³, Frank J. Gonzalez³, and David E. Moller¹.
¹ Department of Molecular Endocrinology, Merck Research Laboratories, Rahway, New Jersey.
² Department of Safety Assessment/Genetic and Cellular Toxicology, Merck Research Laboratories, West Point, Pennsylvania.
³ Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Methods: measurement of CYP4A levels. CYP4A protein levels were assessed in whole liver homogenate, and samples were solubilized in 0.5% sodium cholate/Triton X-100, using rabbit anti-rat CYP4A1 as the primary antibody (Gentest Corp., Waltham, MA) and goat anti-rabbit Ig, horseradish peroxidase-conjugated, as the secondary antibody (Pierce Chemical Co., Rockford, IL). Solubilized samples were diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6, to give approximately 20 ng of protein/50 µL. This was adsorbed to 96-well Maxisorp titer plates (Nalge Nunc International, Rochester, NY) for 1 hour at 37 °C. Wells were then rinsed four times with 0.05% Tween 20 in Ca2+- and Mg2+-free PBS (rinse/block buffer), and 100 µL of primary antibody diluted 1:4000 in Superblock (Pierce Chemical Co.) containing 0.05% Tween 20 (antibody dilution buffer) was added to each well. After 1 hour of incubation at 37 °C, plates were rinsed as above with rinse/block buffer, 100 µL of secondary antibody diluted 1:2000 in antibody dilution buffer was added, and plates were incubated for 1 hour at 37 °C. After incubation, plates were rinsed as above, 100 µL of ABTS 1 STEP (Pierce Chemical Co.) was added, and plates were incubated at room temperature for 20 minutes. The reaction was stopped by addition of 50 °C 2% SDS containing 0.5 mg/mL Proteinase K, and plates were read immediately in a SpectraMax 340 plate reader (Molecular Devices, Corp.)

caspase-3

The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer’s disease-associated neurodegeneration and oxidative stress

J. Neuroscience 20: 7345-52 (2000).

Isabell Greeve¹, Irm Hermans-Borgmeyer¹, Claire Brellinger¹, Dagmar Kasper¹, Teresa Gomez-Isla², Christian Behl³, Bodo Levkau⁴, and Roger M. Nitsch⁵.
¹ Center for Molecular Neurobiology Hamburg, University of Hamburg, 20246 Hamburg, Germany.
² Department of Neurology, Clinica Universitaria de Navarra, Pamplona 31008, Navarra, Spain.
³ Max Planck Institute for Psychiatry, 80804 Munich, Germany.
⁴ Institute for Arteriosclerosis Research, University of Münster, 48149 Münster, Germany.
⁵ Division of Psychiatry Research, University of Zurich, 8008 Zurich, Switzerland.

Methods: caspase-3 activity. Caspase-3 activity was measured in cell lysates of EGFP clones plated to identical densities by using the caspase-3 assay kit (PharMingen, Becton Dickinson GmbH). After exposure to 200 µM H2O2 for 2 and 4 hours or to 25 µM A25-35 for 4 hours, cells were washed briefly in PBS and lysed in 100 µL 10 mM Tris-HCl, pH 7.5, 10 mM NaH2PO4/NaHPO4, pH 7.5, 130 mM NaCl, 1% Triton-X-100, 10 nM NaPPi. Lysates (100 µg protein) were incubated in 200 µL HEPES buffer for 1 hour at 37 °C with 5 µg of the caspase-3 fluorogenic substrate Ac-DEVD-AMC or with 5 µg Ac-DEVD-AMC in the presence of 0.5 µg of the caspase-3 aldehyde inhibitor Ac-DEVD-CHO in a 96-multiwell plate. AMC liberated from Ac-DEVD by caspase cleavage was measured on a spectrofluorometer (Gemini, Molecular Devices Corp.) at excitation wavelength of 380 nm and an emission wavelength spectrum from 420 to 460 nm.

Fluorometric protease assays in the Gemini microplate spectrofluorometer: example using caspase-3

Molecular Devices MaxLine Application Note #35 (1999).

Evelyn McGown, Ph.D. and Anna Lam, B.S.

Summary. This application note describes how to optimize a protease assay in the Gemini microplate spectrofluorometer. We chose to use the protease caspase-3 and a fluorogenic peptide substrate containing a coumarin derivative. Fluorogenic peptide substrates can interfere with the measurement of their own hydrolysis products. If the emission spectra overlap, the assay may have a high background. If the absorption spectra overlap, or if the absorbance of the substrate overlaps with the emission of the product, the substrate can quench the signal from the product. Therefore, both the excitation and emission wavelengths must be optimized to minimize interference from the substrate, while maintaining sufficiently high product fluorescence. The Gemini microplate spectrofluorometer facilitates the process by having dual monochromators that allow optimal excitation and emission wavelengths to be easily determined.

Methods: caspase-3 assay. Z–DEVD–AMC substrate was purchased as part of Molecular Probes EnzChek Caspase-3 Assay Kit #1 (catalog #E-13183). Recombinant caspase-3 enzyme was purchased from Calbiochem (catalog #235417), and 7-Amino-4-Methylcoumarin (AMC) from Sigma (catalog #A 9891). Black 96-stripwell plates and white 96-well plates were purchased from Corning Costar. All dilutions of the substrate and enzyme were made with 1X Reaction Buffer (prepared by diluting 200 µL of the 5X Reaction Buffer and 5 µL of DTT, both included in the kit, with 795 µL of deionized water.) Stock Z-DEVD-AMC (10 mM) was prepared by adding 520 µL of DMSO into the vial of Z-DEVD-AMC. Working solutions of Z-DEVD-AMC stock were typically 1:100 dilutions of the stock, i.e. 5 µL of the stock solution into 495 µL of 1X Reaction Buffer. The stock enzyme was frozen in 2 or 4 µL aliquots (to avoid repeated freeze/thaw cycles), and working enzyme solutions were prepared by diluting the stock 1:10 with 1X Reaction Buffer. The stock AMC solution was 2 mM in DMSO. The optimal instrument settings are as follows: Ex 368 nm/Em 467 nm with either a 420 nm or 435 nm emission cutoff filter. The estimated limit of detection using white microplates was ~0.02 ng per well and for black microplates was 0.10 ng per well.