A fluorogenic substrate for detection of organophosphatase activity

A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.     

An improved fluorogenic substrate for the detection of alkaline phosphatase activity

We designed a new alkaline phosphatase (ALP)-sensitive fluorogenic probe in which a self-immolative spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic compound to improve substrate specificity. Enzymatic hydrolysis converts the fluorogenic substrate 1 to a highly fluorescent reporter 3, thus allowing for the fast and quantitative analysis of ALP activity with greatly increased affinity for the enzyme.     

Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods

In the current work, a synthesis of new sensitive fluorescence substrates of cathepsin G is reported. The substrate sequence was selected using combinatorial chemistry methods. The starting structure of chromogenic cathepsin G substrate Ac-Phe-Val-Thr-Gnf-ANB-NH(2), where Gnf stands for 4-guanidine-l-phenylalanine, was modified by replacing the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence donor. An amide of amino benzoic acid (ANB-NH(2)) was used as an acceptor. This peptide, exhibiting effective fluorescence resonance energy transfer (FRET) phenomena, was used as a starting structure to construct the library Mca-Phe-Val-Thr-Gnf-X(1)-X(2)-ANB-NH(2), where in both variable X positions all proteinogenic amino acid residues except Cys were introduced. Deconvolution of such a library, performed by the iterative method in solution, revealed prime site preferences of cathepsin G. Finally, the most susceptible sequence, Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH(2), was selected. The determined value of the specificity constant (k(cat)/K(M) = 252 x 10(3)M(-1)xs(-1)) was two orders of magnitude higher than that obtained for the parent compound. By the use of this substrate, we were able to detect as little as 70 pM of the enzyme studied.     

A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells

A derivative of rhodamine 110 has been designed and assessed as a probe for cytochrome P450 activity. This probe is the first to utilize a 'trimethyl lock' that is triggered by cleavage of an ether bond. In vitro, fluorescence was manifested by the CYP1A1 isozyme with k(cat)/K(M)=8.8x10(3)M(-1)s(-1) and K(M)=0.09microM. In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol.     

6-Aminoquinoline as a fluorogenic leaving group in peptide cleavage reactions: A new fluorogenic substrate for chymotrypsin

A new fluorogenic substrate capable of measuring the amidolytic activity of chymotrypsin and based upon the enzyme-catalyzed release of a highly fluorescent aromatic amine, 6-aminoquinoline, was prepared. The substrate, 6-(N-glutaryl-l-phenylalanylamido)quinoline, was found to have at pH 8.0 and 25°C K_m = 1.77 mm and k_cat = 1.4 × 10^?1 s^?1. The aminoquinoline is a unique leaving group in that its appearance can be measured fluorometrically at its excitation and emission maxima, while, under these conditions, fluorescence associated with unhydrolyzed substrate is negligible.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.     

Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).     

Sensitive fluorogenic substrate for alkaline phosphatase

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.     

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity

ABSTRACT

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method’s high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.     

N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins.

A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.004 mM, kcat = 104 s-1, and those for subtilisin BPN' are Km = 0.020 mM, kcat = 49 s-1. The substrate is extremely sensitive for subtilisins; the specificity constants are 10-fold higher than the corresponding values for the widely used substrate, succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, and 200- to 1000-fold higher than the values obtained with succinyl-Ala-Ala-Phe-4-nitroanilide. The favorable effect of the anthraniloyl group as a P4 residue in the substrate sequence Ala-Ala-Phe-4-nitroanilide was assumed to be due to an ability to stiffen S4-P4 interactions. The mechanism proposed is hydrogen bond formation between the phenol group of tyrosine-104 and the amino group of the anthraniloyl moiety. In the spectrophotometric assay with the new substrate, the lower detection limit for subtilisin Carlsberg was 1 nM.     

A new fluorogenic substrate for chymotrypsin.

The action of pepsin on a dilute solution of skim milk produces a sigmoidal increase in the turbidity of the solution. The time course of the increase depends on the amount of pepsin present. Turbidity changes can be measured in a recording spectrophotometer, providing an automated form of the milk clotting assay, which will easily determine 20 ng of pepsin.     

A new fluorogenic substrate for human galactocerebroside-beta-D-galactosidase

A new fluorogenic compound--6-hexadecanoylamino-4-methyl-umbelliferyl-beta-D-gala cto pyranoside (HMGal), a substrate for human galactocerebroside beta-D-galactosidase (HG), has been synthesized. A method for determining the HG activity based on the use of HMGal as a fluorogenic substrate has been developed. The specificity of HMGal hydrolysis by HG has been demonstrated in experiments with enzyme preparations from human skin fibroblasts and leukocytes in normally and in hereditary glycolipidosis (GM1-gangliosidosis and Krabbe's disease). The use of HMGal permits to markedly increase the sensitivity of the method used for determining the HG activity.     

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.     

A fluorogenic substrate for the continuous assaying of aryl sulfatases

The most common fluorogenic substrate for assaying aryl sulfatases (ARSs) is 4-methylumbelliferyl sulfate (MUS). However, ARSs operate optimally at pH values that are less than the pK(a) (7.8) of the reaction product of MUS, 4-methylumbelliferone (4-MU). Thus, a major disadvantage of this assay is that it is usually run in a discontinuous mode due to the need for basification of the reaction mixture to achieve complete ionization of the phenolic products and maximum fluorescence. To circumvent this problem, 6,8-difluoro-4-methylumbelliferyl sulfate (DiFMUS) was prepared and examined as a substrate for ARSs. The product of the reaction is 6,8-difluoro-4-methylumbelliferone, a known coumarin with fluorescent properties equal to those of 4-MU, and has a pK(a) of 4.9. This allowed for the continuous assaying of human placental ARSs A, B, and C, which operate optimally between pH 5.0 and pH 7.0. Furthermore, DiFMUS exhibited a lower K(m) (up to 20-fold) for the ARSs than did MUS; for ARSA and ARSB, it exhibited a greater V(max) than did MUS. This substrate should have considerable utility for the continuous assay of ARS activity.     

An FET-type charge sensor for highly sensitive detection of DNA sequence

We have fabricated an field effect transistor (FET)-type DNA charge sensor based on 0.5 microm standard complementary metal oxide semiconductor (CMOS) technology which can detect the deoxyribonucleic acid (DNA) probe's immobilization and information on hybridization by sensing the variation of drain current due to DNA charge and investigated its electrical characteristics. FET-type charge sensor for detecting DNA sequence is a semiconductor sensor measuring the change of electric charge caused by DNA probe's immobilization on the gate metal, based on the field effect mechanism of MOSFET. It was fabricated in p-channel (P) MOSFET-type because the phosphate groups present in DNA have a negative charge and this charge determines the effective gate potential of PMOSFET. Gold (Au) which has a chemical affinity with thiol was used as the gate metal in order to immobilize DNA. The gate potential is determined by the electric charge which DNA possesses. Variation of the drain current versus time was measured. The drain current increased when thiol DNA and target DNA were injected into the solution, because of the field effect due to the electrical charge of DNA molecules. The experimental validity was verified by the results of mass changes detected using quartz crystal microbalance (QCM) under the same measurement condition. Therefore it is confirmed that DNA sequence can be detected by measuring the variation of the drain current due to the variation of DNA charge and the proposed FET-type DNA charge sensor might be useful in the development for DNA chips.     

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.