Scanning the prime-site substrate specificity of proteolytic enzymes: a novel assay based on ligand-enhanced lanthanide ion fluorescence

A novel method for assaying the substrate specificity of proteolytic enzymes has been developed utilizing ligand-enhanced lanthanide ion fluorescence. This approach was used to develop peptide libraries to probe substrate specificity in the prime sites of proteolytic enzymes. A positional scanning synthetic combinatorial library of fluorogenic peptides was synthesized and used to determine the extended prime site specificity of bovine -chymotrypsin. The enzyme showed a preference for Lys and Arg in the P1′ position, rather broad specificity in the P2′ position, and a slight Arg specificity in the P3′ position. The specificity profile of bovine -chymotrypsin agrees well with previously reported data, and the substrate library reported herein should provide valuable information about the prime site substrate specificities of other proteolytic enzymes as well. Furthermore, the continuous fluorogenic assay described may prove useful in analyzing the activity of other hydrolytic enzymes.     

Application of spectrophotometric methods to the determination of protein bound to agarose beads.

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.     

A method to detect proteinase activity using unprocessed X-ray films.

Routine assays to detect proteinases in biological samples are generally tedious and time-consuming. To expedite the recognition of proteinases, we have developed an assay utilizing the gelatin on the surface of an unprocessed Kodak X-Omat AR film as the proteolytic substrate. A positive reaction is indicated by a clear zone on the film after it has been rinsed with running water. This proteinase assay has been found to be inexpensive, rapid, and simple. Besides its ease of use, this assay has been found to be quantitatively reproducible with a well-defined endpoint. More importantly, this assay method is applicable to a variety of proteolytic enzymes under diverse pH (5-8.5) and salt conditions (up to 5 M NaCl) and has a sensitivity similar to that of azocoll. Since the assay does not require sophisticated equipment, it is useful as a general laboratory procedure.     

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.     

An o-phthalaldehyde spectrophotometric assay for proteinases

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.     

Isolation and properties of enzyme preparations from the whale pancreas

A simple and convenient technique was developed for isolation of the proteolytic enzyme complexes from the whale (Balaenoptera) pancreas. The proposed techniques enables the proteolytic complexes to be obtained with the protein yield 2.6 times higher than the classical procedure. The proteolytic activity increased 3.2 times (casein as a substrate), esterase activities, 1.4 times (N-benzoyl-L-tyrosine methyl ester as a substrate) and 1.2 times (N-alpha-benzoyl-L-arginine ethyl ester as a substrate). Soybean and barley trypsin inhibitors and ovomycoid in free and immobilized state inhibit the esterase activities of the proteolytic complexes. An additional purification of the proteolytic complexes was carried out using the affinity sorbent Soybean trypsin inhibitor--Sepharose 4B. The molecular weight of the enzymes determined by means of PAAG electrophoresis was found to be 20 000-20 500. The hydrolysis of some synthetic substrates by the proteolytic enzyme complexes obtained according to the proposed techniques was being studied.     

Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.     

Determination of proteolytic enzymes by flow-injection analysis

Quantitation of proteolytic enzymes using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide has been adapted to flow-injection analysis. This procedure has been developed using two different proteases: subtilisin and chymotrypsin. For both enzymes the influence of substrate concentration on spectrophotometric response has been studied. The assay is based on the merging zones technique combined with a washing step. Results are obtained in less than 15 s and samples may be run at a rate of 90/h with good reproducibility. A linear relation between peak heights and enzyme concentrations was observed for 0-0.15 Anson unit/liter of subtilisin and for 0-30 mg/liter of a commercial preparation of chymotrypsin. The method requires only small sample volumes, and the consumption of the chromogenic substrate is reduced to a minimum by using intermittent pumping.     

A microassay for proteolytic activity

A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions from column chromatography.     

A microassay for proteases using succinylcasein as a substrate.

A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is demonstrated.     

A convenient automated method for the determination of proteolytic enzymes

An automated method for the assay of proteolytic enzymes is described. The insoluble dye-protein complex, Remazolbrilliant Blue-hide powder, is used as the substrate and is readily digested by trypsin, elastase, subtilisin, thermolysin, chymotrypsin, and to a lesser extent pronase, pepsin, and bacterial collagenase. The proteolytic activity of crude microbial culture preparations is expressed in terms of an equivalent concentration of crystalline trypsin, which itself can be readily determined within the concentration range 0.25–5.0 μg/ml.     

Demonstration of a cell-associated, inactive precursor of an exocellular protease produced by Pseudomonas aeruginosa

The enzymatically active form of protease 1, the major exocellular protein produced by Pseudomonas aeruginosa strain 34362, has been shown to exist exclusively exocellularly with no significant cell-associated activity. However, the presence of a cell-associated, enzymatically inactive protein which is serologically cross-reactive with, and convertible to, active enzyme has been demonstrated. One method of conversion of "precursor" to active enzyme is via limited proteolysis. Two assay systems for precursor were developed, one a radioimmune assay, and the other a proteolytic activation procedure. Localization studies suggest that the association while more tenacious than classical periplasmic enzymes is still an ionic rather than a covalent one. Kinetics of production studies showed to precursor to be synthesized early in the growth cycle and to accumulate prior to the rapid release of the active enzyme. Molecular weight studies showed only slight changes produced upon activation.     

Separation of E. coli tRNA-Tyr precursor RNA from E. coli tRNA by hydroxyapatite chromatography

An assay procedure is described for triosephosphate isomerase based on measurement of the ellipticity of l-glyceraldehyde 3-phosphate remaining when d,l-glyceraldehyde 3-phosphate is the source of substrate and d-glyceraldehyde 3-phosphate is converted by triosephosphate isomerase to dihydroxyacetone phosphate. The assay method has advantages over the conventional coupled-enzyme assays in that it circumvents the difficulties posed by instability of the coupling enzymes and their cofactors, as well as by inhibitors of triosephosphate isomerase which may be present in preparations of the coupling enzymes. Although the method is not suited for routine assays during purification or in most clinical applications, it has advantages for detailed kinetic studies where pH, temperature, or other factors cause the coupled-enzyme assay procedures to be unreliable.     

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 simple linear relationship and definition of a unit for proteinase activity

A mathematical transformation involving the $\text{3}\text{2}$ power of the number of milligrams of soluble nitrogen released from the substrate makes possible the expression of proteolytic activity in terms of the quantity of enzyme required to cause a given change in the substrate. The relationship is linear for several different enzymes including papain, patent flour, malted barley, malted wheat flour, and several fungal preparations acting on either Bacto-hemoglobin or gluten substrate.One unit of proteinase activity is defined as that activity contained by a quantity of enzyme-active material which gives an increase in soluble nitrogen in a 10-ml. aliquot from the filtrate corresponding to the intersection of the straight line with the transformed value representing 1 ml. of 0.0714 N alkali.This method of expressing proteolytic activity greatly simplifies the expression of proteolytic activity and has been found convenient for analytical purposes as well as for comparing activities of different enzymes acting on the same substrate.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.     

A peptidyl derivative of [3H]aniline as a sensitive, stable, protease substrate.

A peptidyl derivative of [³H]aniline, Gly-Gly-Arg-[³H]anilide, can be used as a substrate in a convenient and sensitive assay procedure for trypsin, urokinase, and plasminogen activator from transformed cells. The extent of hydrolysis can be determined simply by selective extraction of the product [³H]aniline into an organic phase containing a scintillant. (The uncleaved peptide is not appreciably soluble in this phase and is not counted.) The reaction is of comparable sensitivity to fluorimetric assays, but has the advantage that no cleanup of the biological sample is required, since it is far less subject to interference from fluorescence quenching. Other peptidyl anilides should be useful for assaying proteolytic enzymes with widely varying specificities.     

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.     

A new, rapid, and sensitive assay for adenosine 5'-phosphosulphate (APS) kinase.

A new method for the determination of adenosine 5′-phosphosulphate (APS) kinase activity using a spectrophotometric assay is described. This procedure involves the spectrophotometric determination of sulphate- or APS-dependent production of ADP in the presence of pyruvate kinase and lactate dehydrogenase. Methods are described that overcome interference from contaminating enzymes and compounds. This method also provides a means for a critical examination of the substrate specificity of the sulphate-activating enzymes.