Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes.

The design and application of a recently developed type of fluorogenic substrates for proteolytic enzymes is reviewed. The substrates consist of peptide chains constructed to match the specificity of the particular enzyme and to bear a suitable chromophore at each side of the cleavable bond. One of the chromophores is a fluorescent group and the other is a quencher that causes a great reduction of fluorescence intensity of the fluorophore, either by direct intramolecular encounter or by radiationless resonance energy transfer. Enzymic cleavage of the molecule is followed by release of fluorescence as the result of cancelling the quenching interaction between the chromophores. The properties of such substrates and their possible future applications are discussed.     

Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.     

A study of aspartyl proteases using intramolecularly quenched fluorogenic peptide substrates

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Y = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.     

A study of aspartyl proteases using intramolecularly quenched fluorogenic peptide substrates

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded or (-pNa in place of -Ded), where X=Ala, Ala-Ala, or Val-Ala and Y=−, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.     

A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging

There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.     

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.     

New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.

A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in peptides with the general structure: DABCYL-Lys-Phe-Gly-Xxx-Ala-Ala-EDANS. The substrates were used to evaluate the effect of amino acid structure in the S1' position on the kinetic parameters for papain catalyzed hydrolysis.     

New fluorogenic substrates for renin

A simple and sensitive fluorometric assay was developed to test renin activity within several hours. Two new fluorogenic peptides, Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (octapeptide-MCA) and a succinyl derivative of the octapeptide-MCA were synthesized and used as a renin substrate. Renin cleaved the substrates at the Leu-Leu bond, releasing Leu-Val-Tyr-MCA. Three amino acids of this product were then successively split off by the auxiliary enzyme, leucine aminopeptidase, to liberate free 7-amino-4-methylcoumarin (AMC). The generation of the fluorescent 7-amino-4-methylcoumarin was proportional to renin concentrations up to 100 mGoldblatt U/tube. The optimal pH of renin reaction for both substrates was 6.5 to 7.0. As low as 5 mGoldblatt U of renin could be detected by this method. This method was applied to the assay of renin during its purification.     

New dipeptide fluorogenic substrates of human tissue kallikrein]

Based on 4-methylcoumarinyl-7-amide (Amc) arginine and a series N-alkyloxycarbonyl derivatives of phenylalanine, eleven Amc-derivatives of the type ROCO-Phe-Arg-Amc (R = alkyl) were synthesized; also were n-C3H7OCO-Leu-Arg-Amc and n-C3H7OCO-D-Phe-Arg-Amc synthesized. The enzymatic hydrolysis of these compounds under the action of tissue and plasma human kallikreins were studied. Tissue kallikrein from human urine hydrolyzed the compounds with R = n-propyl and n-butyl and n-C3H7OCO-Leu-Arg-Amc more readily than the known substrates Z-Phe-Arg-Amc and H-Pro-Phe-Arg-Amc. n-C3H7OCO-D-Phe-Arg-Amc is a weak inhibitor of this enzyme (Ki = 1.5.10(-4) M). Human plasma kallikrein hydrolyzed these novel substrates at a lower rate than Z-Phe-Arg-Amc.     

Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates

A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2, 4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K(m) for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was approximately 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.     

Hydrosoluble fluorogenic substrates for plasmin.

New hydrosoluble fluorogenic substrates for plasmin gluconoylpeptidyl-3-amido-9-ethylcarbazole were synthesized. The substitution of the N-terminal end of the peptides by a gluconoyl group prevents the substrates from aminopeptidase degradation and highly increases their hydrosolubility. The substitution of the peptide C-terminal end by a 3-amino-9-ethylcarbazole group leads to substrates suitable for direct fluorometric assay of plasmin present in cell supernatants or in cell lysates. On the basis of the kinetic parameters of the substrate hydrolysis by plasmin, it was found that D amino acids in the P2 position decrease systematically the kinetic constants of the substrates. The L configuration of the P2 amino acid appears therefore as essential in optimum substrates for plasmin.     

New fluorogenic peptide substrates for plasmin

Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.     

New fluorogenic substrates for N-arginine dibasic convertase

N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The characterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2, 4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectroscopy. The rate of hydrolysis of the fluorogenic substrates is proportional to enzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic parameters for the fluorescent peptides (Km values of approximately 1.0 microM, and Vmax values of approximately 1 microM/(min. mg) are similar to those obtained with peptide hormones as substrates.     

A general and direct method for the solid phase synthesis of intramolecularly quenched fluorogenic protease substrates using a bifunctional coumarin derivative

A general method for the solid phase preparation offluorogenic peptide substrates or intramolecularly quenchedones (IQFS) is presented, using the highly fluorescentbifunctional coumarin derivative 7-amino-4-coumarinyl-acetic acid. The key feature of this method is theconjugation of H–Aca–OH through its carboxyl group on theresin, followed by the development of the peptide chainthrough its amino group, using standard Fmoc-derived solidphase peptide synthesis methodology. The 2,4-dinitrophenylgroup was used as quencher and introduced directly to theresin-bound peptides. The IQFSDnp–Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (2) andfour Dnp–X-Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (3–6), where X = Val, Lys, Ser and Glu at P₆ position,potential substrates for cathepsin D, were synthesized forproving the utility of the method. The compoundsH–Ile–Lys–Leu–Aca–OH (7),H–Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (8),H–Leu–Aca–OH (9), Dnp–Leu–Aca–OH (10) and Dnp-Leu-OH (11) were also synthesized for comparisonpurposes. The fluorescence properties of compounds 9and 10 were measured.     

Novel fluorogenic substrates for acid phosphatase.

Fluorinated versions of fluorescein diphosphate (FDP) can provide significantly enhanced fluorescence upon hydrolysis by acid phosphatase, as compared with FDP, when measured at the reaction pH.     

Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins

Human plasma low density lipoproteins (LDL) contain one major apoprotein of apparent Mr = 550,000 designated apolipoprotein B-100 (apo-B-100) and in some LDL preparations, minor components termed apo-B-74 (Mr = 410,000) and apo-B-26 (Mr = 145,000). The structural and metabolic relationships among these LDL apoproteins remain obscure. In the present study, we show that the mixing of proteolytic inhibitors with blood at the moment of collection prevents the appearance of apo-B-74 and -26 in plasma LDL indicating that these peptides are derived by proteolytic degradation of apo-B-100. In order to simulate the degradation in vitro, LDL were digested with plasmin, trypsin, chymotrypsin, thrombin, and tissue and plasma kallikreins and the degradation products analyzed by polyacrylamide gradient gel electrophoresis. While plasmin, trypsin, and chymotrypsin caused extensive degradation of apo-B-100, thrombin, and tissue and plasma kallikreins generated limited cleavage patterns. LDL digested with thrombin contained stoichiometric amounts of two peptides with apparent Mr = 385,000 and 170,000. Mixing experiments showed that the thrombin-derived peptides of apo-B-100 did not co-migrate with apo-B-74 and B-26 during electrophoresis indicating that these peptides were different. In contrast, LDL digested with kallikrein contained stoichiometric amounts of two peptides with apparent molecular weights identical to apo-B-74 and -26. Together, the above results indicate that apo-B-74 and -26 are degradation products of apo-B-100 and are not produced by the action of thrombin. Whether the expression of a kallikrein-like activity in vivo accounts for the specific degradation of LDL B-100 to yield LDL B-74 and -26 remains to be determined.     

One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry

We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.     

Attenuated kinin release from human neutrophil elastase-pretreated kininogens by tissue and plasma kallikreins

Components of kinin-forming systems operating at inflammatory sites are likely to interact with elastase that is released by recruited neutrophils and may, at least temporarily, constitute the major proteolytic activity present at these sites. The aim of this work was to determine the effect of kininogen degradation by human neutrophil elastase (HNE) on kinin generation by tissue and plasma kallikreins. We show that the digestion of both low molecular mass (LK) and high molecular mass (HK) forms of human kininogen by HNE renders them essentially unsusceptible to processing by human urinary kallikrein (tissue-type) and also significantly quenches the kinin release from HK by plasma kallikrein. Studies with synthetic model heptadecapeptide substrates, ISLMKRPPGFSPFRSSR and SLMKRPPGFSPFRSSRI, confirmed the inability of tissue kallikrein to process peptides at either termini of the internal kinin sequence, while plasma kallikrein was shown to release the kinin C-terminus relatively easily. The HNE-generated fragments of kininogens were separated by HPLC and the fractions containing internal kinin sequences were identified by a kinin-specific immunoenzymatic test after trypsin digestion. These fractions were analyzed by electrospray-ionization mass spectrometry. In this way, multiple peptides containing the kinin sequence flanked by only a few amino acid residues at each terminus were identified in elastase digests of both LK and HK. These results suggest that elastase may be involved in quenching the kinin-release cascade at the late stages of the inflammatory reaction.