Assay Of Proteolytic Activity Research Articles

Ethylene Effects on Amylase Activity from Isolated Barley Aleurone Layers

The effect of protease inhibitors on the response of gibberellic acid-treated barley aleurone layers to ethylene was examined. In the absence of protease inhibitors, ethylene plus gibberellic acid initially increased the production of amylase activity relative to layers incubated with gibberellic acid alone. Exposure to ethylene plus gibberellic acid for 48 hours or longer, however, led to depressed levels of amylase activity compared to samples incubated with gibberellic acid in hydrocarbon-free air.The direct assay of proteolytic activity revealed a small increase in activity in response to ethylene. The significance of this response was probed further by including inhibitors of barley proteases in the incubation medium. When potassium bromate was introduced, ethylene did not cause any alteration in amylase activity compared to samples incubated in hydrocarbon-free air. However, in the presence of N-ethylmaleimide, ethylene treatment induced a 52% increase in amylase activity recovered from samples after 48 hours. These results suggest that proteases contribute to the loss of amylase activity in response to ethylene and thus alter the apparent effect of ethylene on amylase synthesis. The effect of protease inhibitors on other hydrolases is also discussed.During the incubation period, the pH of the medium declined significantly. However, ethylene had no effect on the extent of this decline.

Assay of proteolytic activity by ultrafiltration

Assay of proteolytic activity by ultrafiltration

Assay for proteolytic activity using a new fluorogenic substrate (peptidyl-3-amino-9-ethyl-carbazole); quantitative determination of lipopolysaccharide at the level of one picogram.

A new sensitive fluorimetric assay has been developed using peptidyl-3-amino-9-ethyl-carbazole as substrate. The fluorescence intensity of free 3-amino-9-ethyl-carbazole (AEC) at 460 nm is between two and three orders of magnitude higher than the fluorescence intensity of acyl-AEC. The release of AEC from a peptidyl derivative by proteases may be monitored continuously during the hydrolysis step or may be quantified upon addition of a general inhibitor such as benzamidinium chloride. Using N-benzoyl-arginyl-AEC as substrate, as little as 1 ng trypsin may be detected. Using t-butyloxycarbonyl-Val-Leu-Gly-Arg-AEC and the amoebocyte lysate of Limulus polyphemus, as little as 1 pg lipopolysaccharide can be detected. This fluorimetric method allows detection of trace amounts of lipopolysaccharide (endotoxins) in various biological materials, including sera.

A direct simultaneous plate assay of proteoglycan and collagen degradation by cells in culture and its application to synovial cells.

1. A radiochemical plate assay is presented that allows a simultaneous evaluation of the capacity of cells in culture to degrade proteoglycan and collagen. Its principle consists of monitoring the release of soluble radioactive degradation products from Multiwell culture plates coated with dried reconstituted 3H-labelled-proteoglycan/14C-labelled-collagen mixed gels. The plates can also be used for the assay of proteolytic activities within enzyme solutions. 2. When cultured on the plates, rabbit synovial cells degrade collagen and proteoglycan almost simultaneously, owing to the secretion of collagenase and of a proteoglycan-degrading metal-dependent neutral proteinase.

Specificity of the BT-PABA test for the diagnosis of exocrine pancreatic insufficiency in the dog.

Exocrine pancreatic insufficiency in the dog has been assessed by the oral administration of the synthetic peptide N-benzoyl-L-tyrosyl-p-aminobenzoic acid (BT-PABA), a specific substrate for pancreatic chymotrypsin. The subsequent assay of PABA in either the plasma or the urine clearly differentiated control animals from those with exocrine pancreatic insufficiency (EPI), the results being unaffected by combination of this pancreatic function with a xylose absorption test. Possible interference with the specificity of the peptide test for the diagnosis of EPI was examined in six animals with small intestinal disease. In a group of four animals, with features resembling chronic tropical sprue in man, the results were comparable to those of the control group. In the fifth case, however, the results were indistinguishable from those of the EPI group, the estimation of sodium PABA absorption and the assay of proteolytic activity in the duodenal juice demonstrating that this was due to defective hydrolysis of the peptide. In the sixth case, diffuse intestinal lymphosarcoma and a marked villous atrophy were associated with an apparent reduction in the absorption of sodium PABA. However, although the plasma PABA concentrations following oral BT-PABA were subnormal, they were distinctly higher than those of the EPI group. These findings suggest that small intestinal abnormalities do not affect PABA absorption sufficiently to interfere with the specificity of the peptide test for the detection of severe EPI in the dog. This insufficiency may occasionally be secondary to small intestinal disease.

Determination of proteolytic activity using l-[4,5-3h]leucine-labelled globin as a substrate

A method is described for the assay of proteolytic activity, based on the digestion of L-[4,5-3H]leucine globin. L-[4,5-3H]Leucine was incorporated into the substrate at the stage of haemoglobin biosynthesis, using rabbit erythrocytes. Assay methods for proteolytic enzymes have been based on the digestion of haemoglobin, serum albumin or casein, and the determination of the trichloroacetic acid-soluble products [1,2]. More sensitive methods have been developed by using haemoglobin labelled with a fluorescent [3-5] or radioactive marker [6,7]. These methods avoid the errors which beset the Anson procedure, such as interference by impurities (purines at 280 nm and reducing compounds at 700 nm) [8]. However, methods using labelled proteins as a substrate present a number of problems, the most troublesome of which are the high blank values and the use of non-physiological substrates when chemically modified proteins are employed. In the present communication a simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5-3H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products.

A fluorescamine-based sensitive method for the assay of proteinases, capable of detecting the initial cleavage steps of a protein.

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of 'specific' proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N-amino-terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a) the accumulation of N-amino termini in excess over that of the intact substrate; (b) a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed.

Evidence for an alkaline protease in vaccinia virus

Purified vaccinia virions (Lister strain) were found to contain a proteolytic activity that can use exogenous proteins as a substrate once the virions are disrupted at pH 10.6. Assays of proteolytic activity were carried out with labeled cellular proteins bound to an insoluble support. Purified viral cores obtained after Nonidet-P40 and 2-mercaptoethanol treatment of virus retained the activity. Temperature and pH dependence were observed as well as an inhibitory effect of heavy metal ions.

QUANTITATIVE ASSAY OF SPECIFIC PROTEOLYTIC ACTIVITY IN FOOD PROTEIN HYDROLYZATES BY SINGLE RADIAL IMMUNODIFFUSION

ABSTRACTThe increasing use of proteolytic enzymes in food production has established the need for a quantitative and sensitive assay for proteolytic activity in protein foods. The classical methods are not suitable because of the presence of large amounts of proteins and peptides. A quantitative procedure based on single radial immunodiffusion was developed and found to be applicable to the determination of subtilisin Carlsberg activity in protein hydrolyzates. Less than 0.5 ppm subtilisin Carlsberg could be detected in a 7% soy protein hydrolyzate. With minor modifications the method should be applicable to many of the other enzymes used in food processing.

Assay of proteolytic activity by gelatin liquefaction (trypsin assay by gelatin liquefaction)

A new assay for proteolytic enzymes and their inhibitors based on the liquefaction of gelatin gels has been developed. The assay is more sensitive than colorimetric tests, can be carried out upon colored or turbid samples and does not require the use of a spectrophotometer. The procedure consists of incubating the test sample with a fluid gelatin solution, cooling the solution so that it sets to a firm gel and then incubating at a warmer temperature until the gel liquefies. The time taken for liquefaction is several days for a sample of pure buffer, about one minute for a sample containing 0.5 Mg of trypsin per ml and longer for samples containing less trypsin, following an empirical calibration. An appreciable decrease in tryptic activity can be detected in the presence of only 0.1 KIU of Trasylol® (bovine pancreatic trypsin inhibitor) by this method.

Sensitive, solid-phase assay of proteolytic activity

Insolubilized, radioodinated proteins are used as substrates for the assay of proteolytic activity. Trypsin hydrolysis of Sepharose-bound 125I-labeled casein, hemoglobin, immunoglobulin G, and bovine serum albumin (specific activities of 1–10 μCi/μg) is described to illustrate the sensitivity and applicability of this procedure. Over the course of a 5-min assay period, the activity of 0.1–5 μg of trypsin/ml is quantitated. Since 50 μl is used as the assay sample, this procedure is capable of detecting the equivalent of 5 ng of trypsin.

2] Some sensitive methods for the assay of trypsinlike enzymes

There is a need for more sensitive assays for proteolytic enzymes as increasing numbers are discovered that are present in minute quantities and have biologically important roles. Among these, serine proteases in particular predominate, since they are essential in blood clotting, clot removal, complement action, and fertilization, to name a few areas of current interest. Most of the individual enzymes have a trypsin-like specificity. This chapter details some of the sensitive methods used for the assay of trypsin-like enzymes. The chapter also discusses active site titration, reagents or fluorimetric enzyme titration and rate assays-like fluorometric esterase assays, plasminogen activator assay, isotopic TsArgOMe assay and fluorometric assay of proteolytic activity.

32] Leucostoma peptidase A

Publisher Summary Leucostoma peptidase A is the most abundant protease in the venom of the western cottonmouth moccasin (Agkistrodon piscivorus leucostoma) from which it can be isolated in good yield and a high degree of homogeneity. This endopeptidase possesses some characteristics similar to those of bacterial neutral proteases, in as much as it is inhibited by metal complexing reagents, is a metalloprotein that contains calcium and zinc, and has a substrate specificity dictated primarily by the identity of the residue contributing the amino group to the susceptible peptide bond. This chapter deals with an assay procedure which states that Leucostoma peptidase A readily digests casein and denatured hemoglobin, either of which can be used as a substrate to assay the proteolytic activity of fractions obtained during enzyme isolation. It explains the hemoglobin assay for proteolytic activity, arylamidase assay. The chapter also discusses the enzyme isolation, properties such as purity, stability, physical properties, metal content, pH optimum and substrate specificity.

The automated kinetic determination of enzyme activities and constituents of plants. 3. Determination of bromelain (author's transl)

With bromelain as protease model a kinetic method, capable of automatization, is described for the assay of proteolytic activities. The method uses milk protein as a substrate. The volume of the test mixture is fixed to 1 ml; the readings are performed at 340 nm. With the bromelain preparation used (2 mAnson-U/mg) a linear decline of O.D. (deltaA/min) between 5 and 50 mug bromelain/ml is observed. Other proteases as well as protease inhibitors may be analyzed by the same method also.

Direct fluorimetric assay for proteolytic activity against intact proteins

The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates resulting in a decline in fluorescence. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates in this assay procedure. The activity of 1 μg chymotrypsin or trypsin and 100 ng pepsin could be easily detected by this method within 4 to 8 min depending upon the protein substrate. The digestive enzymes and bromelain exhibited activity against most if not all six of the protein substrates used. In contrast, the extract from germinating pumpkin seeds exhibited significant activity only against pumpkin seed globulin, with maximal activity at pH 7.4. Compared with the assay method for proteolytic activity utilizing ninhydrin analysis of the reaction products, this method was at least 10 times more rapid and gave significant detectable activity with much lower quantities of proteolytic enzyme.

Assay for lipolytic and proteolytic activity using marine substrates.

Nondestructive assay procedures for determining microbial lipolytic and proteolytic activity on marine substrates were developed and tested with 287 isolates of bacteria, filamentous fungi, and yeasts. A definite substrate specificity was noted when the enzymatic activities on marine and nonmarine substrates was compared. Of 170 lipolytic isolates, 14 were only active on menhaden oil, 11 could hydrolyze menhaden oil and Tween 80 and/or tributyrin, and 145 isolates could only hydrolyze one or both of the nonmarine lipids. Of the 198 proteolytic isolates, 10 were specific for codfish extract, 152 were active against the marine substrate plus casein and/or gelatin, and 36 were specific for nonmarine substrates.

A sensitive assay for proteolytic enzymes using bacterial luciferase as a substrate

A general assay for proteolytic enzymes using bacterial luciferase as a substrate is described. This luciferase is rapidly inactivated by unusually small amounts of different proteases having a wide spectrum of specificities. The activity is lost exponentially; the pseudo-first-order rate constant for inactivation is proportional to the amount of protease. Since luciferase activity can be measured by a very simple and rapid assay, it affords an accurate, sensitive, and convenient assay for proteolytic activity. This technique is capable of detecting as little as 20 ng of trypsin, requires no centrifugation, and is not hampered by the presence of contaminating pigments in the protease preparation. It is compared at pH 6, 7, and 8 to the phenol color assay and the azocoll assay using seven different proteases.

Extracellular Protease of Penicillium roqueforti. I. Production and Characteristics of Crude Enzyme Preparation1,2

The proteolytic activity of various strains of Penicillium roqueforti (ATCC 10110, ATCC 6987, Japan BP-13) was qualitatively assayed by streaking a suspension of spores on the surface of Czapek-Dox agar containing 1.0% sodium caseinate and 20mM calcium chloride. Extracellular proteolytic activity was based on the precipitation of the calcium-sensitive caseins in the agar medium. Protease was produced by shake-culturing (225rpm) the various strains of fungi in Czapek-Dox broth containing .5% Proteose-Peptone No. 3 at 25 to 27C for 72 to 78h. A cell-free extract of the strain releasing the largest quantities of extracellular protease (BP-13) was prepared by centrifugation of the vegetative growth, followed by filtering the supernatant through Millipore filters. The assay for proteolytic activity was based on the amount of trichloroacetic acid-soluble nitrogen released after 9min incubation at 30C with 1.0% Hammersten casein at pH 5.75. The extracellular protease had pH optima of 3.0 and 5.5 for bovine serum albumin and casein. Maximum stability to pH occurred in the range of 3.0 to 6.0. The optimum temperature for proteolytic activity was 46C; thermal inactivation commenced at 48C.

A fluorescent assay for proteolytic enzymes

A method is described which permits the assay of proteolytic enzyme activity on protein substrates without precipitation or filtration steps, utilizing a fluorescent reagent which is specific for primary amines. The assay is about 100 times more sensitive than the Lowry method, much faster and less complicated. Ambiguities concerning the absorbing species are largely eliminated. The reagent (Fluorescamine, Hoffmann-La Roche RO-20-7234) yields fluorescent compounds with amino acids at pH 9.0 and with peptides at pH 6.8, but possesses no fluorescence by itself.

Inactivation of alpha- and beta-chymotrypin by intact Hymenolepis diminuta (Cestoda).

When specimens of intact Hymenolepis diminuta were incubated in the presence of α- or β-chymotrypsin, assays for proteolytic activity following removal of the worms showed an inactivation of both enzymes. The amount of inactivation in either case was dependent upon the enzyme concentration, total number of worms (total worm weight) present, period of time worms were incubated with the enzymes, and pH of the assay medium. Inactivation of α- and β-chymotrypsin was independent of available surface area and the presence of polyions, was irreversible, and ceased upon removal of the worms from the medium. Intact worms also inactivited the zymogen, α-chymotrypsinogen A. The data suggest that the inactivation of α- and β-chymotrypsin resembles that previously reported for inactivation of trypsin by intact H. diminuta.