Lowlevel protein preparation for characterization by mass spectrometry

When attempting low-level (i.e. sub-picomole) protein identification or sequencing, a number of precautions have to be taken in order to avoid sample contamination and losses. All buffers and reagents should be made fresh using chemicals of the highest available purity. Stocks of chemicals should be kept in a clean environment. Dust is a common source of contamination and every effort should be made to clean thoroughly all equipment used in sample handling (e.g. microcentrifuge tubes, gloves,...

Detection of proteases using unprocessed Xray films8 Equipment and reagents

X-Omat XAR 5 X-ray film from Kodak Controlled-temperature incubator Corporation (several sizes are available) 1 Take a sheet of unprocessed X-ray film. Add 50 p.1 drops of different dilutions of a protease in an appropriate buffer,1 and incubate the film at 42 C for 1 h. 2 Wash film gently under running tap water for 1 min. 3 Dry and visually determine clearing of film. Adapted from (53). ''Film is not compatible with buffers containing guanidine hydrochloride (3m) or potassium thiocyanate (1...

Kinetically controlled synthesis

Studies on the kinetically controlled synthesis of condensation products such as oligosaccharides (13), peptides (14) and p-lactam antibiotics (3) using hydrolases as catalysts have presented kinetic evidence that the nucleophile NH must be bound to the acyl- (or glycosyl-) enzyme before it can deacylate this intermediate. The reactions determining the yield of the condensation product from the acyl-enzyme are shown in Figure 3. This mechanism also applies for other condensation products....

Solvent deuterium isotope effects

Solvent deuterium isotope effects can be used in two ways. First, the catalytic activity should be measured in pure D20 to determine if there is an effect of the substitution of deuterium for hydrogen on the catalytic process. If protons are transferred in the rate-limiting step of the mechanism, the rate will be reduced by a factor of two to three. This observation is diagnostic of a general acid- or general base-catalysed process and is expected for enzymes of the second, non-nucleophilic...

Determination of primary specificity of a protease

There are two general ways to explore the specificity of a new enzyme. The first is to examine the cleavage pattern of the activity on a standard substrate such as the insulin B-chain. This sequence contains a useful variety of bonds available for cleavage. The products are small peptides that can readily be separated by HPLC, and a considerable literature exists documenting the properties of the products of digestion by the common proteases. Incubation of insulin B-chain with the newly...

Membrane ProX carboxypeptidase 5556

Synonyms carboxypeptidase P, microsomal carboxypeptidase Primaiy specificity -PrP (P cannot be Ser, Gly) Suggested conditions for use pH 3.7-5.2 stabilized by non-ionic detergents, Triton X-100, Tween Inhibitors PCMB, iodoacetic acid, DFP, SDS, diethylpyrocarbonate, fatty acids Notes a renal brush border membrane exopeptidase stabilized at low protein and acidic pH values by Triton X-100

Contents

1 Proteolytic enzymes nomenclature and classification i 2 Terminology and nomenclature l Peptidase and related terms 1 Specificity-subsite terminology 3 Catalytic type 3 Homology 3 3 The EC classification of peptidases 4 What information does the EC list contain 6 When and how can a newly-discovered peptidase be added to the EC list 4 The MEROPS system for the classification of peptidases 12 Individual peptidases 17 Uses of the MEROPS system 38 5 Steps one might take on discovering a new...

YGluX carboxypeptidase 4547

Synonyms -y-glutamyl hydrolase, carboxypeptidase G, conjugase, folate conjugase, pteroyl-poly-y-glutamate hydrolase, lysosomal y -glutamyl carboxypeptidase Primary specificity Pj-Pi (Pj C-terminal amino acid, Pj 7-glutamic acid) Source Pseudomonas sp Substrate pteroyl triglutamate with C-terminal Glu carrying a 14C label Specific activity 0.5 Ci mol Stability storage store at -0 C (the human form of carboxypeptidase G requires zinc for stability) Suggested conditions for use 33 mM sodium...

Introduction

A landmark in the development of any field of study is the appearance of a sound system of nomenclature and classification for the objects with which it deals. The introduction of the Linnaean system for naming and classifying organisms in the eighteenth century and the invention of a system of nomenclature for enzymes in the 1950s were such key events, and their value has been obvious. Both nomenclature and classification are vitally important for information-handling, allowing people to...

Fluorimetric assays

Fluorimetric assays employ either the inherent fluorescence of an amino acid or depend on the fluorescent properties of products. In such assays the spectro-fluorimeter has to be standardized with known concentrations of the fluorescent substance, or standardized using secondary fluors. The choice of fluor is dependent on the type of substrate being utilized and the sensitivity necessary for the experiments. For example, experiments designed to evaluate kinetic parameters of an enzyme will...

J S Bond

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, Hersbey, PA17033-0850, USA In 1967, Schechter and Berger (1 introduced a system of nomenclature to describe the interaction of proteases and their substrates which is widely used in the protease literature. In this system, the binding site for a polypeptide substrate on a protease is envisioned as a series of subsites each subsite interacts with one amino acid residue of the substrate. By convention, the...

Reversible inhibitors

An equilibrium between the complexed form of an enzyme and free enzyme characterizes reversible inhibition. Sometimes, a reversible inhibitor may bind so tightly that it is not possible to measure a binding constant. Under this condition the inhibitor is sometimes classified as 'pseudo-irreversible' and it is then appropriate to analyse it as an irreversible inhibitor. For freely reversible inhibitors, provided that I > E x 10, the fraction of free enzyme existing in equilibrium with a...

Conventional purification

In the absence of affinity purification, conventional purification procedures are used. To establish effective purification steps of an inhibitor it is important to determine the specific activity of the inhibitor sample after each purification step. When the inhibitor concentration is very high, almost complete inhibition of the enzyme activity is detected. In this case, the sample must be diluted to give only 25-75 inhibition of the enzymatic activity to determine the specific activity (see...

Spectrophotometric assays

Spectrophotometry assays rely on the difference in molar absorptivity between the substrates and products. Substrates and products with large differences in molar absorptivities in the visible range are more desirable than those absorbing in the UV range, where absorbance due to enzymes and components of crude extracts can cause interference. In an interesting paper, Cathers and Schloss (20) describe an enzyme-coupled assay for proteases. Peptide and amino acid products of protease hydrolysis...

Prediction of nicksites

There is considerable value in a prediction method that could identify sites of limited proteolysis in a known protein. It would allow rational design of the protein in order to increase or decrease its overall proteolytic susceptibility that is typically correlated with its thermal stability (18). There have been few attempts to do this. Simple correlations with loops and (J-turns have been considered (19,20), but for restricted protein subsets. A more general approach has been developed to...

P and Pi specificity

For the synthesis of a peptide bond PfPr (Appendix I) it is necessary to use a protease with a high - or Pj-specificity for the amino acid that should be added to a given Pi or Pi amino acid. Data on the P Pj specificities of proteases, based on extensive studies on hydrolysis of different substrates, have been compiled (21-24). The Pj specificity in peptide synthesis is given by the ratio (kx kH)app. For Pj as a nucleophile in the kinetically controlled synthesis of Pi-Pi, (kr Wapp increases...

Preface

In the decade since the first edition of this book there has been a dramatic expansion in the identification and structural characterization of proteolytic enzymes complemented by knowledge of the functional and regulatory aspects. We have an increasing understanding of mechanisms and the critical roles that proteases play in developmental, physiological, and pathological processes. The information explosion concurrent with the sequencing of multiple genomes, including the human genome, will...

Method

1 Prepare a saturated solution of HCCA in acetone. Vortex briefly and centrifuge to remove insoluble material. 2 Prepare a solution of nitrocellulose (lOg l) in 1 1 (v v) acetone-isopropanol. 3 The matrix solution is prepared by mixing four volumes of HCCA solution with one volume of nitrocellulose solution. 4 Deposit 0.3 ,1 of matrix solution onto the MS probe. The solution will spread out (no confining edges) and evaporate within a few seconds to produce a thin film. 5 Deposit 1 jul of 10...

Reverse zymography for detection of matrix metailoproteinase inhibitors

Washing buffer 50 mMTris-HCl(pH 7.5), Incubation buffer 50 bim Tris*HCl 5 mm CaC2, 5 am ZnC2. 0.02 NaN3, 2.5 (pH 7.5), 5 mM CaC2, 5 jim ZnC2. 0.02 1 Make a 10 SDS-PAGE gel containing 0.8 mg ml of gelatin (final conc.) and 0.5 g ml (final) of purified proMMP-2 (progelatinase A) or 6 U ml (final activity in the gel 1U digests 1 xg gelatin at 37 eC in 1 min) of gelatinase A or B solution (e.g. conditioned-medium from cultured fibroblasts), It is not necessary to preactivate progelatinases, since...

Aspartic proteases

The best characterized aspartic proteases from mammals (pepsin, chymosin, cathepsin D and renin) are all inhibited by pepstatin A, a pentapeptide-like compound secreted by Streptomyces species, which contains two residues of the unusual amino acid statine acid , K values range from 4.5 x 1011 m for pepsin to 5 x 10 7m for renin, and the original pepstatin structure has been modified in many ways to increase selectivity for different aspartic proteases (11). Renin is an extremely selective...

Methods

1 Equilibrate substrate and enzyme solutions at 25 C (see Table 2, note b). 2 Pipette 250 jxl of substrate into a 1.5 ml microcentrifuge tube. Initiate the assay by pipetting 150 enzyme into the substrate. Mix the reaction gently, but thoroughly, Incubate the reaction mixture at 25 C for a set time period (15-30 min)6. Prepare a reagent blank by replacing the enzyme solution with buffer in the above procedure. It is advisable to ran each sample in duplicate or triplicate. 3 Terminate the assay...

Solution conditions

Native state proteolytic reactions require reaction conditions that are compatible with two native proteins the proteinase and the substrate. The pH of the digestion mixture is able to affect the catalytic activity of the proteinase through active-site ionizations, and the structure of the proteinase and the substrate. The optimal pH for the proteinase may not be the same as that for maximum stability or relevant functional state of the substrate. However, the pH optima of most proteinases are...

Notes on ideal assays

Other chapters in this volume describe assays for the detection and analysis of proteolytic enzymes. Exacting studies of the mechanism of an enzyme require a well-characterized assay system. In particular, the substrate must be pure and stable (see Table 2), at least throughout the course of exposure to enzyme. The products of attack by the enzyme must also be fully characterized. Ideally, there should be one or more readily identifiable differences between the substrate and the cleavage...

The use of mutants

For the isolation of protein from microorganisms, mutants deficient in one or more proteinase offer a particularly effective means of eliminating some proteolytic problems. Mutants have been isolated in studies concerned with the physiological function of proteinases, but their availability can be of considerable value in studies of other proteins from the same organism. This is exemplified by the yeast proteolytic system for which mutants lacking one of four proteinases (yscA, yscB, yscD and...

Protein identification by MALDI peptide mass mapping

Several features of MALDI-MS make this technique an excellent tool for the initial analysis of peptides derived from in-gel digestions. First, sample preparation is very simple but nonetheless highly efficient. Homogeneous matrix surfaces, necessary for obtaining high mass accuracy and low femtomole sensitivity (HCCA, sinnapinic acid and ferulic acid) can easily be prepared by the fast evaporation method described in Protocol 5 (35,36). The addition of nitrocellulose to the matrix (33,37)...

Cysteine proteases

Given their broad specificity for papain family members and fast-reacting mechanism the trans-epoxysuccinyl peptides are almost ideal active-site titrants. Com- pound E-64 is used to titrate these cysteine proteases primarily because of its ready availability. The procedure, taken from Barrett ft a . (44), is applicable to the lysosomal proteases cathepsins B, H, L and S. and the plant proteases papain, chymopapain, ficin and bromelain, but not clostripain it is carried out as described in...

Endopeptidase and aminopeptidase substrates

The simplest endopeptidase substrate consists of a doubly blocked aminoacyl residue in which the acyl group is amide-bonded to a group that yields a chromophore upon hydrolysis. A typical substrate is Bz-L-Arg-NHNan. A corresponding aminopeptidase substrate will be L-Arg-NHNan. The amino-terminus of an amino acid or peptide can be blocked with any of a number of reagents, of which those used in peptide synthesis predominate some common examples of NH2 blocking groups are Bz, Z, Ac, and Sue (see...

Chemical modificationidentification

Because the serine and cysteine proteases both possess strongly nucleophilic catalytic residues, the covalent chemical modification of those enzymes using group-specific reagents is a reasonable way to establish the mechanistic class of the enzyme. The classic example is the use of Dip-F to chemically inactivate the serine proteases. This reaction relies on the enhanced reactivity of the catalytic serine residue in those enzymes. Chymotrypsin possesses 33 serine residues, but the active-site...

Protease inhibitors in cell culture

Many of the forgoing procedures can be adapted from purified systems to cell culture (Table 6). The key is to use inhibitors that are cell-penetrating and stable to culture conditions. Often, these inhibitors are methylated to block acidic moieties and improve cell penetration and, whenever possible, it is preferable to use the methylated derivative. The methylated compounds are frequently inactive in vitro, and it is assumed that cellular esterases demethylate them to the active inhibitor....

Coagulation factor Xa 3134

Synonyms factor XA , thrombokinase, prothrombase, prothrombinase EC Number EC 3.4.21.6 Type serine endopeptidase Primary specificity P2-Arg-Pi- (cleaves between Arg and PJ Pt usually Gly Pi non-specific but He and Thr preferred) Source bovine, human plasma Substrate Bz-Ile-Glu-Gly-Arg-4-Na, 25 C Specific activity 1.3 U mg protein, enzyme stabilized with bovine serum albumin Stability storage stable at 4 C Suggested conditions for use pH 8.3, 25 C Inhibitors antithrombin III (with heparin), DFP,...

Example reaction schemes

A single proteolytic cleavage, in which neither substrate nor product interferes with the proteolytic reaction, should obey pseudo first-order kinetics. For example, the cleavage of a protein ab into fragments a and b. The general equation describing all types of simple, first order, proteolytic reactions is Where At is the measured property at time t, A0 defines the property at the start of the reaction, Af the property at the end, and k is the rate constant for proteolysis. For example, a...

References

Methods Enzymol. 244,126. 2. Kobayushi, K. and Smith, J. A. (1987). J. Biol. Chem. 262,11435. 3. Jones, W. M., Scaloni, A., and Manning, J. M. (1994). Methods Enzymol. 244, 227. 4. Tanaka, N., Takeuchi, M. and Ichishima, E. (1977). Biochim. Biophys. Acta 485, 406. 5. Shintani, T., Kobayashi, M., and Ichishima, E. (1996). J. Biochem. 120, 974. 6. Suzuki, K., Imajoh, S., Emori, Y Kawasaki, H Minami, Y., and Ohno, S. (1987). FEBS Lett. 220, 271. 7. Suzuki, K.,...

Preventing proteolysis by denaturation

If proteins of interest are not required in an active form, as in the case of total cell protein analysis by SDS-PAGE, the most direct means of preventing proteinase activity is to use strongly denaturing conditions either during or immediately following cell disruption. Protein extraction can be carried out in the presence of urea, SDS or guanidinium hydrochloride. However, proteinases are often more resistant to denaturation than other proteins, and mild denaturation conditions may accelerate...

Endopeptidase K

Synonyms proteinase K, Tritirachium alkaline proteinase EC Number EC 3.4.21.64 Type serine endopeptidase Primary specificity -PrPi- (P non-specific but aromatic or hydrophobic amino acids preferred) Specific activity 10-300 U mg protein Stability storage stable at 4 C. Stored in 50 mM Tris-HCl, pH 8.0, containing 1 mM CaCl2 stable for months at 4 C Suggested conditions for use pH 7.5-12 Notes a homologue of subtilisin enzyme is capable of cleaving native proteins, used to release nucleic acids...

Cm1 mm

Figure 6 Small-scale peptide purification. Peptides obtained by in-gel proteolytic cleavage are concentrated and purified using a small amount of POROS R2 resin. First, the sample is applied to a microcolumn as shown on the right and washed with a few mlcrolitres of 5 formic acid. The column is then transferred into the capillary holder and aligned with the nanoelectrospray needle. Finally, peptides are eluted into the nanoelectrospray needle with two 0.5-p.l aliquots of 50 methanol in 5 formic...

Experimental considerations

Many factors determine the nature of a specific protein-proteinase interaction. The interaction is so dependent upon the particular molecular recognition events intrinsic to the process that exact guidelines are impossible. Nevertheless, some general guiding principles can usually be applied. In particular, the potential manipulation of limited proteolysis experiments is greatly enhanced if the kinetics are understood. To a first approximation, proteolytic reactions, especially those that are...

Peptide mass mapping

Protein identification by peptide mass mapping is based on the idea that while any one of a list of experimentally determined peptide masses might be found in a number of proteins, it is very unlikely that many peptide masses would all be found within the same protein by chance (23-27). In this approach, a list of peptide masses is generated by proteolytic degradation of a protein followed by MS (peptide mass mapping). These measured peptide masses are compared with a theoretical protease...

E64 1314

Target proteinases cysteine proteinases Molecular weight 357.4 Effective concentration 1-10 xM. Stable for days at neutral pH Stock solution 1 mM in aqueous solution. Stable for months at -20 C Notes E-64 is an effective irreversible inhibitor of cysteine proteinases that does not affect cysteine residues in other enzymes or reacts with low molecular weight thiols such as 2-mercaptoethanol. It is an excellent active-site titrant

PH dependence of the kinetic parameters

The next step will then be to determine Km and kcat values over the whole range of pH where activity can be observed and where the enzyme is stable. Construction of buffered solutions for this purpose should be done according to the suggestions of Ellis and Morrison (42). Their recommendations are designed to avoid the large changes in ionic strength normally encountered when buffers of different pH are prepared using a single buffer species. Following collection of sufficient data at each pH...

ArProteinase inhibitor

Target proteinases trypsin, plasma proteinases, elastase, most mammalian serine proteinases Effective concentration equimolar with proteinase Stock solution stable for several months in solutions containing 0.01 NaN3. Can be stored at - 80 C but should not be refrozen. Unstable below pH 5.5 Notes inactivated by some non-serine proteinases. Inactivated by oxidation of active site methionine residue

Choice of proteinase

The most commonly used proteinases are trypsin, lysine specific proteinases (endoproteinase Lys-C and lysyl peptidase), endoproteinase Glu-C and endo-proteinase Asp-N. These proteinases have a restricted primary specificity and have proven useful for producing peptides for sequence and other analyses. Less-specific proteinases may give many peptides that are too small to give much data. Amino acid analysis can help choose a suitable proteinase by showing how often a particular amino acid, e.g....

Methods for determining the mechanistic class

Describing the detailed mechanism of a protease can range from a crude classification based on susceptibility to a group of protease inhibitors (PMSF, E-64, pepstatin, and o-phenanthroline) to an elegant discussion of stereo-electronic factors based on high-resolution X-ray ciystallography. Between these extremes there are numerous experimental protocols for obtaining data on the mechanistic grouping of a new activity. Among these are response to a broader range of inhibitors, chemical...

Proteases in peptide synthesis limitations and perspectives

For practical purposes, more peptides are synthesized by chemical peptide synthesis than in protease-catalysed processes. For some peptides a combination of chemical and enzymatic condensation steps is used (9,10). The automation of chemical peptide synthesis using peptide synthesizers, and newer developments that reduce the amounts of problematic solvents, still favour this procedure over the enzymatic approach. However, the latter has advantages, including pre vention of racemization during...

Sample desalting and concentration for nanoelectrospray MS analysis

Nanoelectrospray capillaries (custom made, or commercially available from Protana A S, Danmark) Custom-made capillary holder (Figure 6) Table micocentrifuge (e.g. PicoFuge, Statagene) 1 Mount a microcolumn (i.e. a nanoelectrospray needle) in a custom-made holder as outlined in Figure 6. 2 Prepare a slurry of POROS R2 re versed-phase resin in methanol and remove fines by repeated agitation settling of the slurry with subsequent removal of the super-natant.b 3 Pipette 5-7 m-1 of the slurry into...

Determination of mechanistic class of a protease

Serine protease inhibitor 100 mM PMSF in DM SO (prepare fresh) Cysteine protease inhibitor 10mM E64 in water (stable at -20 C) Aspartic protease inhibitor 100 jig ml pepstatin in DMSO (stable at -20 C) 1 To six tubes, each containing 0.98 ml of buffer, add 10 jjlI protease, and incubate at 30 Cfor5min. 2 To separate tubes, add 10 jxl of each inhibitor (tubes 1-4), 10 xl of DMSO (tube 5) or 10 xl of buffer (tube 6). 3 Incubate all six tubes at 30 C for 10-60 min. 4 Initiate the protease assay...

Finding purification and characterization of natural protease inhibitors

Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, Hammersmith, 1 Aspenlea Road, London W6 8LH, UK The Burnham Institute, 10901 North Torrey Pines Road, La Jolla CA 92037, USA The activity of a proteolytic enzyme in a living organism is precisely regulated by synthesis and secretion of the enzyme, by zymogen activation and frequently by inhibition. Although some proteinase activities may be limited by veiy restricted substrate selectivity, or by specific cellular...

Metal chelators

Advantages reversible some selectivity can be achieved for zinc peptidases Disadvantages can inactivate many other enzymes and proteins. Metalloproteases contain a zinc ion that participates in catalysis by polarizing a water molecule to attack the substrate-peptide bond. This differentiates them from proteases such as the calpains and some trypsin-likc serine proteases, whose activities are stabilized by, but not necessarily dependent on, the presence of calcium. Both the zinc-dependent...

The serine peptidases

This is the most thoroughly studied class of enzymes in the protease field, and perhaps in all of enzymology. Our current understanding of the mechanism of serine peptidases is illustrative of the whole group of enzymes involved in fragment transfer (1), although in this case the transfer is to water. The intermediate transfer of the acyl portion of a substrate to form a covalent bond with a functional group of the enzyme is the common feature between the serine peptidases and other...

Introductionthe importance of mechanistic classification

The opening chapter of this volume has outlined the four mechanistic classes serine and cysteine proteases (those that form covalent enzyme complexes) and aspartic and metalloproteases (those that do not form covalent enzyme complexes). This distinction into two major mechanistic groups is of profound consequence since the strategy for inhibition is totally different for these two classes. Those enzymes of the first general class have strongly nucleophilic amino acids at their catalytic site....

Cell disruption and fractionation

The distribution of cellular proteolytic enzymes among different compartments means that careful disruption of the cells followed by subcellular fractionation can yield samples which are low in endogenous proteolytic activity. Tissues should be disrupted so as to avoid breakage of proteinase-rich vacuoles and lysosomes. This requires the use of gentle techniques (homogenization, freeze-thaw in liquid nitrogen and the omission of detergents such as Triton X-100 which dissolve vacuolar lysosomal...

Peptidase assay for electrophoretically separated enzymes using synthetic substrates

Substrate 0. 1-1.0 mg ml aminoacyl- or N-blocked amino acyl-NHNap in the buffer of choice Staining solution Fast Garnet GBC (2 mg ml in water) Prepared fresh just prior to use Assay buffer the choice of buffer will depend on the enzyme being studied examples are phosphate, Tris-HCl, Tricine and HEPES. Buffers should be at least 1 Fractionate enzyme samples by poly aery lam ide gel electrophoresis under either native or denaturing conditions.'b 2 Remove the gel from glass plates or tubes, and...

Achromopeptidase

Synonyms lysyl endopeptidase, API, Achromobacter proteinase I EC Number EC 3.4.21.50 Type serine endopeptidase (chymotrypsin analogue) Primary specificity Pi-Pi (Lys is preferred for Pi) Source Achromobacter lyticus M497-1 Substrate denatured casein, Boc-Val-Leu-Lys-AMC Specific activity 4 U mg Stability storage stable at pH 4.0-10.0 and can be stored at -20 C in 1-100 mM Tris-HCl buffer at pH 6-10 at enzyme concentrations higher than 0.1 mg ml Suggested conditions for use 50 mM Tris, pH 9.0,...

Analysing the data by nonlinear curve fitting

The data set derived from a simple proteolytic reaction will be a set of (t, At) data pairs to which will be fitted an equation describing the behaviour. All first-order reactions are described by equations that are non-linear. This does not mean that the line of At as a function of t is curved, but that the independent variable has a non-linear relationship to the parameters of the equation in this instance, At (at any one t value) does not vary linearly according to the value of the rate...

Endoproteinase ArgC

Synonyms submandibular proteinase A, esteroprotease EC Number no assigned EC number Type serine endopeptidase Primary specificity -Arg-P -Source mouse submaxillaris gland Substrate Tos-Arg-OMe, pH 8.0, 37 C Specific activity 100-225 (imol min.mg protein Stability storage stable dry at 4 C in solution at -20 C Suggested conditions for use 0.1 m NH4C03, pH 8.0-8.5, 25-37 C Inhibitors DFP, TLCK, Hg2+, Cu2+, Zn2+, a2-macroglobulin Notes the enzyme loses specificity during long incubations...

Second Edition

Department of Veterinary Preclinical Sciences, University of Liverpool, U.K. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, U.S.A. Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide in Athens Auckland Bangkok Bogot Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence...

Strategies for limited proteolysis experiments

The key factor in implementing the experimental design for a limited proteolysis experiment is the need to ensure that proteolysis only occurs within the course of the reaction. The design of the experiment must ensure that the proteinase only acts during the incubation period. If kinetic data are required, it is also essential that the activity of the proteinase does not change throughout the reaction, and thus, autolysis or inactivation must be prevented. In the absence of any preliminary...

Coelution of peptides

A typical proteolytic digestion produces many peptides, some of which co-elute. Decreasing the number of peptides produced is one way to decrease this problem, which is a reason to choose one proteinase, e.g. lysyl peptidase instead of trypsin, for a first digestion of a protein. The problems to be addressed are how to determine if a fraction from a chromatography run contains more than one peptide, and then how to separate them. The shape of a peak can be a clue that a fraction contains more...

Aspartic Peptidase

Figure 3 (A) Schematic representation of the general acid-general base catalytic mechanism of the aspartic peptidase type of enzyme. Breakdown of the tetrahedral intermediate gives a product complex containing both halves of the substrate, and this scheme indicates that dissociation of either can follow to give an acyl product complex or an amino product complex. (B) Schematic representation of the multitude of hydrogen -bonding interactions available in the active-site cleft of a typical...

The Merops system for the classification of peptidases

As we have seen, the EC system places each of the peptidases in one of just 13 sub-subclasses, and only five of the sub-subclasses are available for the very numerous endopeptidases (comprising 204 of the total of 281 entries). This inevitably lumps together peptidases that are very different. Most importantly, it takes no account of structural groupings of peptidases that would reflect evolutionary relationships. About 1992, N. D. Rawlings and the present author started to develop a new form...

Glutamyl endopeptidase 4849

Synonyms endoproteinase Glu-C, Protease V8, staphylococcal serine proteinase EC Number EC 3.4.21.19 Type serine endopeptidase Primary specificity P Pa-P Pj-Pi-P (Pi Glu or Asp, Glu preferred Asp preferred at P4 Ala or Val preferred at P3 Phe preferred at P2 Pro disfavoured at P3, P , Pj Asp disfavoured at Pi) Source Staphylococcus aureus V8 Substrate oxidized insulin B-chain digestion (HPLC analysis) CBZ-Phe-Leu-Glu-4-NA, pH 7.8, 25 C (Fluka) N-t-BOC-L-Glu-a-phenyl ester, pH 7.8, 37 C (Sigma)...

Phosphoramidon 416

Target proteinases some metallo-endopeptidases Molecular weight 543.6 Effective concentration 1-10 (jlm. Stable for 1 day Stock solution 1 mM in water. Stable for 1 month at -20 C Notes phosphoramidon is an inhibitor of many bacterial metallo- endopeptidases but few of mammalian origin. Neprilysin is very susceptible to this inhibitor Target proteinases all serine proteinases Synonyms phenylmethanesulphonyl fluoride, phenylmethylsulphonyl fluoride, a-toluenesulphonyl fluoride Molecular weight...

Simulations and modelling

As proteolytic reaction schemes become more complex, exact analytical solutions are progressively harder to derive. An alternative strategy for analysis of such data, based on the simplifying assumption that each reaction is strictly first order, is to use a general modelling package that can find solutions to the Figure 10 Sequential proteolytic reactions. The screenshot is of a simple, sequential proteolytic reaction A B C where the concentrations of A and B are plotted as a function of time....

Terminology and nomenclature

Endopeptidase And Exopeptidase

The term protease appeared in the German literature of physiological chemistry in the latter part of the nineteenth century in reference to proteolytic enzymes, and was used as a general term embracing all the hydrolases that act on proteins, or further degrade the fragments of them. Around 1930, the need to distinguish separate kinds of protease activity began to be recognized and two sets of terms were independently proposed, one in Germany and one in the United States. In Germany, Grassmann...

List of suppliers

395 Page Mill Road, PO Box 10395, Palo Alto, CA 94304, USA Web site www.agilent.com Pharmacia Biotech Biochrom Ltd., Unit 22, Cambridge Science Park, Milton Road, Pharmacia and Upjohn Ltd., Davy Avenue, Knowlhill, Milton Keynes, Buckinghamshire Anderman and Co. Ltd., 145 London Road, Kingston-upon-Thames, Surrey KT2 6NH, UK. Tel 0181 541 0035 Fax 0181 541 0623 Boulevard, PO Box 3100, Fullerton, CA Beckman Coulter UK Ltd., Oakley Court, Kingsmead Business Park, London Road, High Wycombe,...

Endopeptidase assays

Endopeptidase assays can be used to detect a specific enzyme or evaluate a number of enzymes, such as those present in a crude extract. In general, these assays involve the incubation of a protein substrate containing several or many scissile sites of undefined kinetic properties with a protease or a mixture of proteases. Substrates such as gelatin, casein, haemoglobin, etc., are commonly used. Table 1 Common peptidase substrate8 nomenclature substituent-NH-PI-P l'-COO-substituent Table 1...

Protocol list

Autolytic solubilization of dipeptidyl peptidase IV from rat kidney membranes3 28 General scheme for purification of proteolytic enzymes Partial purification of prolyl oligopeptidase EC 3.4.21.26 by acid precipitation and ammonium sulphate fractionation 31 Specialized techniques for proteolytic enzymes Coupling a peptide to an affinity support via an amino group 35 Coupling a peptide to an affinity support via a carboxyl group 36 Purification of the proteasome EC 3.4.99.46 Purification of the...

Activesite titration of cysteine proteases papain family or caspases

Inhibitor titrant, E-64 the unmethylated derivative, also known as E-64C . E-64D is the methylated derivative and is only appropriate for cell culture experiments Substrate, as appropriate for the protease being titrated, and depending on the available method of detection Assay buffer, as appropriate, noting that many papain family enzymes are unstable at neutral pH. Many assay buffers call for detergents such as Brij 35 or Triton X-l00 in the activation buffer the authors recommend...

Standard digestion using iysyi peptidase

100-200 pmole of protein freshly 2 jxg of cytochrome c or other standard protein alkylated using Protocol 2 alkylated using Protocol 2 Lysyl peptidase 1 Add 0.2 g or as appropriate for amount of protein of lysyl peptidase to the alkylated protein, control digestion no substrate and a tube containing the alkylated cytochrome c or other standard protein 3 Add 2 j1 of trifluoroacetic acid to stop the reaction and freeze tubes until analysis A typical ratio is proteinase substrate is 1 100 to 1 30...

Exopeptidase assays

Natural substrates are rarely employed in the routine assay of exopeptidases, because of the diversity of synthetic substrates and the ease with which an elegant assay which uses modified substrates can be developed. Exceptions are exopeptidases involved in the processing of prohormones and other metabolically active peptides. Synthetic substrates are constructed around the chemistry of amino acids and peptides. Novel substituents are added to either the a-NH2 or a-COOH groups of amino acids...

Finding inhibitors by reverse zymography

Some proteinase inhibitors retain inhibitory activity after SDS-PAGli without reduction. Using this principle, Herron et ol. 5 developed a reverse zymography technique to detect tissue inhibitors of metalloproteinase TIMPs , In this technique the sample to be detected is run on SDS-polyacrylamide gel containing a protein substrate copolymerized into the gel. The gel is then incubated with the solution containing matrix metalloproteinases. The procedure was improved by Staskus et al 6 by...

Equipment and reagents

Fluorimeter variable wavelength equipped with temperature-controlled cuvette housing Matched UV grade fluorescence cuvettes examples here employ 1.0 ml cuvettes however, 3 ml cuvettes can also be used by scaling up the procedure Circulating water bath connected to the cuvette housing . Computer or strip chart recorder for monitoring fluorescence output Substrate aminoacyl-NHMec or N blocked aminoacyl-NHMec 0.1-0.5 mM in buffer. Substrate can be diluted to the desired concentration Buffer 50 mM...

The EC classification of peptidases

The internationally recognized scheme for the classification and nomenclature of all enzymes, including peptidases, is that of the IUBMB. The Nomenclature Committee of the IUBMB is the successor to the Enzyme Commission, and the index numbers that it assigns to enzymes are still described as 'EC' numbers 4,10 . The latest full publication of the EC list is in Enzyme Nomenclature 1992 4 , but this has been updated by several supplements relating specifically to peptidases 11-14 . The up to date...

A tool to aid in prediction of sites of limited proteolysis

The prediction program, NICKPRED, has been made available via the internet and can be reached via the URL http sjh.bi.umist.ac.uk nickpred.html Figure 3 . Users may obtain predictions from structures in the current release of the Brookhaven Databank 27 or may submit their own models structures in PDB format. Results are returned via e-mail. The output options of the software allow the user access to the raw, normalized conformational parameter scores, as well as the processed prediction scores...