A new assay for proteases using fluorescent labeling of proteins

A simple assay for proteases based on the fluorescent labeling of insoluble proteins (fibrin) or of soluble casein by 2-methoxy-2,4-diphenyl-3(2H)furanone has been developed. Fluorescence of the liberated peptide-fluorophors resulting from enzymatic hydrolysis is easily measured in the supernatant after separation of the unreacted fluorescent fibrin by centrifugation or from unreacted casein-fluorophor by acid precipitation. Nanogram quantities of trypsin, chymotrypsin, and elastase can be measured.     

A new kinetic assay method for quinone-reducing enzymes

A new kinetic method is described for the assay of quinone-reducing enzymes in various biological materials. It is based on polarographic determination of oxygen uptake in spontaneous oxidation of the diphenol formed as a result of 4-anilino-5-methoxybenzoquinone-1,2 (AMOBQ) enzymic reduction. The stoichiometry of the reducing equivalent transfer in the reaction sequence from NAD(P)H to oxygen has been analyzed. Data are presented on quinone-reducing activity distributions in different tissues.     

A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A

Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.     

A novel and rapid apoptosis assay based on thiol redox status

We present here a novel probe, VitaBright-48, for the evaluation of the cellular level of reduced thiols. Using different cell lines and apoptogenic agents we show that a decrease in the level of reduced thiols correlates with well-known apoptotic markers such as phosphatidylserine translocation and caspase activity. The cell population to be investigated is added to the nonfluorescent stain VitaBright-48, which immediately permeates the cell membrane and reacts with intracellular thiols, forming a fluorescent compound. Quantification of the cell fluorescence directly after staining (without washing) can then be used to determine the population's cellular thiol level at the single cell level. Based on the results presented here, we suggest that measurement of changes in the level of free thiols should be added to the list of phenotypes which may be investigated in order to detect apoptosis.     

A new assay for endocytosis in erythrocyte ghosts based on loss of acetylcholinesterase activity

A new method for assaying endocytosis in erythrocyte ghosts is presented. The method involves measuring the percentage loss of acetylcholinesterase activity which occurs when vacuoles form, making the acetylcholinesterase on the vacuole surface inaccessible. This method is compared to other methods of measuring endocytosis in this system, including phase contrast microscope estimation of vesiculation, stereological analysis of electron micrographs to determine vesiculation and loss of sialic acid accessible to neuraminidase due to endocytosis. Comparison of the percentage loss of acetylcholinesterase activity with the electron micrographic and sialic acid methods showed that all three methods gave a quantitative measure of the percentage of total membrane area taken in as vesicles. Since the acetylcholinesterase method was fast, easy, inexpensive, and quantitative, it was the preferred method for assay of endocytosis. The inhibition of endocytosis by Ca²⁺ was observed with this method; the success of this experiment demonstrated the applicability of the method to the study of inhibitors of endocytosis.     

A new assay for collagenolytic activity.

A new assay procedure for the determination of collagenolytic activity is presented. The substrate can be prepared by simple reduction of the purified acidsoluble rat tail tendon collagen with NaB³H₄. Collagenase activity is determined by measurement of soluble tritiated collagen peptides released. It has proven to be a method with a high degree of sensitivity and reproducibility.     

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.     

A chromogenic assay of substrate depletion by thiol dioxygenases

A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5′-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman’s reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.     

A new spectrophotometric assay for enzymes of purine metabolism. II. Determination of guanase activity.

A new method for the determination of guanase is described. Xanthine, the product of the guanase reaction, is oxidized by xanthine oxidase, forming uric acid and hydrogen peroxide. Hydrogen peroxide is further reduced to water by catalase in the presence of ethanol. The acetaldehyde formed in this reaction step is dehydrogenated NAD or NADP dependent by aldehyde dehydrogenase. The NADH or NADPH production is measured and utilized for the calculation of the guanase activity. The sensitivity of the method can be doubled by the addition of uricase, which oxidizes uric acid to permit the formation of another mole of hydrogen peroxide.     

Stimulatory effect of regucalcin on proteolytic activity in rat renal cortex cytosol: Involvement of thiol proteases

The effect of regucalcin, a calcium-binding protein, on neutral proteolytic activity in the cytosol of rat kidney cortex was investigated. Proteolytic activity was significantly increased by the presence of regucalcin (0. 01-0. 25 M) in the enzyme reaction mixture. This increase was not significantly altered by the addition of CaCl2 (0.01 and 1.0 mM) or EGTA (1.0 mM), indicating that the effect of regucalcin was independent on Ca2+. The effect of regucalcin to increase proteolytic activity was completely prevented in the presence of N-ethylmaleimide (5 mM), a modifying reagent of thiol groups. Proteolytic activity was clearly elevated by dithiothreitol (5 mM). This elevation was further enhanced by regucalcin (0.1 M). Meanwhile, the stimulatory effect of regucalcin on proteolytic activity was not significantly altered in the presence of diisopropylfluorophosphate (2.5 mM), an inhibitor of serine proteases. Also, the regucalcin effect was not appreciably changed by the addition of EDTA (2.5 mM), a chelator of metal ions, indicating that it is not involved in metal-related proteases. These results demonstrate that regucalcin can increase proteolytic activity in the cytosol of rat kidney cortex. Regucalcin may activate thiol proteases independent on Ca2+.     

A new spectrophotometric assay for enzymes of purine metabolism. I. Determination of xanthine oxidase activity.

A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.     

A bioluminescent assay for the sensitive detection of proteases

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.     

A hydrogen peroxide electrode assay to measure thiol peroxidase activity for organoselenium and organotellurium drugs

Molecular mimics of the enzyme glutathione peroxidase (GPx) are increasingly being evaluated as redox active drugs. Their molecular mechanism of action parallels that of the native enzyme; however, a major distinction is that GPx mimics can use alternative thiol substrates to glutathione. This generic thiol peroxidase activity implies that it is necessary to assess a GPx mimic's recognition of a range of cellular thiols in order to determine its potential therapeutic effects. We report an electrochemical assay that, by measuring the rate of decrease of the peroxide substrate, allows the activity of GPx mimics to be directly compared against an array of thiols. The derived pseudo zero-order rate constants, k(obs), for representative GPx mimics range between 0 and 6.6min(-1) and can vary by more than an order of magnitude depending on the thiol electron donor. An additional advantage of the assay is that it enables synergistic interactions between GPx mimics and cellular proteins to be evaluated. Here we report that glutathione disulfide reductase, which is commonly used to evaluate GPx mimic activity, recognizes the GPx mimic ebselen as a substrate, increasing its apparent k(obs). Therefore, reports relying on glutathione disulfide reductase to evaluate GPx mimic activity may exaggerate drug antioxidant action.     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.