A Facile Method to Determine Prolidase Activity Using a Peptide-Specific Fluorometric Reaction.

Prolidase is the only enzyme capable of cleaving imidodipeptides containing C-terminal proline (Pro) or hydroxyproline and plays a crucial role in several physiological processes such as wound healing and cell proliferation. Here, we developed a new method to determine prolidase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal glycine (Gly)-containing peptides using 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA can selectively react with Gly-Pro, the substrate for prolidase, and the prolidase activity is measured by monitoring the decrease in FL intensities. The prolidase activities in fibroblasts and HeLa cells were successfully measured by the proposed method. Compared with classical Chinard's method, our method does not require any caustic acids, pre-incubation to activate the enzyme, and heating for reaction with the detection reagent. The proposed method enables facile and specific measurement for biogenic prolidase activity.

Extremely Simple and Rapid HPLC Analysis of Tocilizumab in Human Serum with Selective Precipitation Using Alkylamine.

An assay using HPLC with fluorescence (FL) detection method for monitoring native FL of tocilizumab (TCZ) in human serum combined with extremely simple and rapid pretreatment without any antigen-antibody reaction was developed. Good separation of TCZ was achieved within 13 min on a Presto FF-C18 column (100 × 4.6 mm i.d., 2 µm). Simple pretreatment with acetonitrile containing primary and secondary alkylamines having longer than C3 in the alkyl chain removed immunoglobulin G subclass 1 and TCZ could be recovered selectively. The spiked calibration curve of TCZ in human serum showed good linearity in the range of 40-1000 µg/mL (r > 0.997). The lower limit of quantitation (S/N = 10) of the TCZ was 19.7 µg/mL. The accuracy was within 103.5-114.9%, and the intra- and inter-day precisions as relative standard deviations were less than 5.3 and 7.8% (n = 5), respectively. The recovery of TCZ was 42.2 ± 3.4% (n = 3). The TCZ in pretreated sample was confirmed to be stable for 6 h (>95%) at room temperature and 24 h (>95%) at 4 °C. The proposed method is considered extremely superior to the previous methods in terms of time requirement for analysis. Therefore, the developed method may be more useful than conventional methods in urgent situations, such as confirming therapeutic efficacy of cytokine-release syndrome by 2019 coronavirus disease.

A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid.

Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was measured using a spectrofluorometer, which reflects the consumption of orotic acid by the OPRT. After optimization of the reaction conditions, the OPRT activity was successfully determined in 15 min of enzyme reaction time without further procedures such as purification of OPRT or deproteination for the analysis. The activity obtained was compatible with the value measured by the radiometric method with [3H]-5-FU as the substrate. The present method provides a reliable and facile measurement of OPRT activity and could be useful for a variety of research fields targeting pyrimidine metabolism.

A Facile Method for the Quantification of Urinary Uracil Concentration by a Uracil-Specific Fluorescence Derivatization Reaction.

A facile and reliable fluorescence method for the quantification of urinary uracil concentration is proposed herein. The assay utilizes a specific fluorescence (FL) derivatization reaction for uracil using 3-methylbenzamidoxime as a fluorogenic reagent. Although the presence of urine inhibited the FL reaction, 10 µL of urine was sufficient for the detection of urinary uracil. The uracil derivative was successfully separated from other fluorescent impurities using simple reversed-phase LC with FL detection. Urinary uracil concentrations from 16 people were compared with the concentrations obtained by the traditional column-switching liquid chromatographic analysis with UV detection. The FL derivative of uracil appeared as a single peak in the chromatograms of all samples. However, several samples showed an additional peak overlapping the uracil peak when using the column-switching method because of UV-active impurities. These results indicated that that the present method is not affected by interfering substances in urine and affords a precise determination of urinary uracil. We expect the proposed method to be applicable for diagnosing dihydropyrimidine dehydrogenase deficiency in 5-fluorouracil chemotherapy.

Simple and rapid analysis of tocilizumab using HPLC-fluorescence detection method.

We developed a novel assay using high-performance liquid chromatography (HPLC) with fluorescence detection for the determination of tocilizumab (TCZ), after it has undergone a facile and rapid pretreatment. TCZ belongs to the same subclass as IgG1 (Immunoglobulin G subclass 1), and we could separate TCZ from IgG1 without antigen-antibody reactions, with the novel detection method. The separation of these antibodies was achieved by pretreatment with an organic solvent containing a base, such as trimethylamine and triethylamine. The effect of these bases on the separation of TCZ is related to the hydrophobicity of the base rather than the electrostatic charge. The results indicated that the surface charge of antibodies changed because of the structural change, even though the difference in the amino acid sequences of the antibodies was very low. Our method is available for the separation of the antibody subclasses, and it would be useful to assay TCZ in blood.

Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction.

Orotic acid is an intermediate in the synthesis pathway of uridine-5'-monophosphate, and increases in body fluids of patients suffering from hereditary disorders such as orotic aciduria and hyperammonemia. In this study, we developed a spectrofluorometric method with or without high-performance liquid chromatography for the selective and sensitive quantification of orotic acid in human biological specimens, using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. This reagent provided intensive fluorescence for only orotic acid amongst 62 compounds including structurally related bio-substances such as nucleic acid bases, nucleosides, nucleotides, amino acids, vitamins, bilirubin, uric acid, urea, creatine, creatinine and sugars. Under optimized reaction conditions, orotic acid was reacted with 4-TFMBAO, K3[Fe(CN)6] and K2CO3 in an aqueous solution. The fluorescence produced from the orotic acid derivative was measured at an excitation of 340 nm and an emission of 460 nm. A concentration of 1.2 µM orotic acid per 1.0 mM creatinine in normal urine and 0.64 nmol orotic acid per 5.0 × 10(5) HeLa cells were determined by this method. The present method permitted the facile quantification of orotic acid in healthy human urine and cultured HeLa cells by spectrofluorometry and/or high-performance liquid chromatography.

A novel fluorescence reaction for N-terminal Ser-containing peptides and its application to assay caspase activity.

Caspases are the key regulatory factors of apoptosis and are also found to be involved in inflammatory cytokinesis. Sensitive and selective determination of caspases has significant importance in evaluation of apoptosis, disease diagnosis, and drug development. Here, we developed an assay method for the determination of caspase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal Ser-containing peptides. FL derivatization of peptides requires heating in the presence of catechol, HEPES buffer (pH 7.5), and sodium periodate. Under optimized conditions, the reaction showed a unique sequence preference for N-terminal Ser-containing peptides, and a lower detection limit (signal/noise [S/N] = 3) of approximately 0.1 µM was obtained for SKTS and SSNSF. Acetylated substrates were enzymatically cleaved to produce N-terminal Ser-containing peptides, which were selectively converted to FL compounds. The enzyme activities were simultaneously determined as low as 2 U (4.3 nM) caspase-3 and 2.5 U (3.3 nM) caspase-8 by high-performance liquid chromatography (HPLC) with FL detection. The proposed assay method does not require any labeled substrates and can be applied to evaluate cell-based apoptosis and also to study apoptosis inhibitors or inducers.

Selective FL quenching or enhancing of diimine ligands by guanine.

Diimine ligand (DL) 1 significantly exhibited the fluorescence quenching upon binding to guanine. Changing at the para-substituent of the phenyl ring from the hydroxyl to bromo groups reversely enhanced the fluorescence in the presence of guanine. The reverse in the fluorescence selectivity indicated the profound effect of the substituent at the para-position of the phenyl ring. The simple synthesis of DL 1 and DL 2 with good selectivity for guanine offers these DLs as promising compounds for chemosensors of other guanine derivatives.

Chemiluminescence detection of telomere DNA in human cells on a membrane by using fluorescein-5-isothiocyanate-labeled primers.

Telomere DNA is related to cell aging and cancer genesis because the telomeric region of DNA sequences at chromosome ends are shortened with cell divisions. Therefore, a sensitive and specific detection method is required for the telomere DNA. Here we propose a chemiluminescence (CL)-based method for the sensitive detection of telomere DNA in human cells. In this study, the telomere DNA was amplified by polymerase chain reaction (PCR) using special forward and reverse primers labeled with fluorescein-5-isothiocyanate (FITC) at the 5' end, and then the FITC-containing PCR products were detected by CL reaction with 3,4,5-trimethoxyphenylglyoxal (TMPG) after electrophoresis followed by Southern blot onto a nylon membrane. The TMPG reagent specifically reacted with guanine moiety in DNA at room temperature and provided CL intensities. The CL intensities from the PCR products could be enhanced approximately 10-fold using FITC-labeled primers as compared with those using nonlabeled primers. The detection limit of the PCR products with the proposed method was 0.3 ng on the membrane. The developed CL method could quantitatively determine the telomere DNA in a small number of human cells (∼350) and gave approximately 10 times higher sensitivity than a conventional fluorescence-based method.

Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane.

In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27-31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14-20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays.

Selective and facile assay of human immunodeficiency virus protease activity by a novel fluorogenic reaction.

A highly selective and facile assay of human immunodeficiency virus protease (HIV-PR) has been required for the screening of medicinal inhibitors and also for classifying the subtypes of HIV in the therapeutic treatment of acquired immune deficiency syndrome (AIDS). This article describes a novel assay method of HIV-PR based on the selective fluorogenic reaction of peptides. A peptide fragment generated from a substrate by the enzymatic digestion with HIV-PR could be selectively quantified by the spectrofluorometric detection after the fluorogenic reaction with catechol in the presence of sodium periodate and sodium borate (pH 7.0). This assay system uses an N-terminal acetyl peptide as the substrate and crude extracts from Escherichia coli expressing recombinant HIV-PR. The activity obtained by the proposed assay correlated with that obtained by a conventional HIV-PR assay based on fluorescence resonance energy transfer detection.

Simultaneous assay for protease activities of hepatitis C virus and human immunodeficiency virus based on fluorescence detection.

Hepatitis C virus protease (HCV-PR) and human immunodeficiency virus protease (HIV-PR) are important for virus maturation, and thus can be used as potential target molecules for the development of antiviral drugs for the treatment of viral infections. In this study, a novel assay was developed to determine HCV-PR activity. This assay is based on a fluorogenic reaction, in which peptide fragments generated from an acetyl peptide substrate by HCV-PR can be selectively converted into a fluorescent derivative, and quantified by high-performance liquid chromatography (HPLC) with fluorescent detection. Herein, several acetyl-peptides can be used as substrates for HPLC. The application of this assay was further validated by simultaneous detection of HCV-PR and HIV-PR in a reaction mixture. The proposed method can differentiate the enzyme activities of HCV-PR and HIV-PR in a sample using their corresponding substrates. The results suggest that this assay can detect various proteases by employing set of substrate peptides under the same reaction conditions.

Fluorescence detection of amino acids in the postcleavage conversions for manual sequencing of a peptide.

A modified Edman degradation method where fluorescent derivatives of amino acids were generated from the postcleavage products of a peptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manual sequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus amino acid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1'-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl-CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ-CIA-amino acids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for amino acids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio=3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manual sequencing of insulin chain B composed of 30 amino acids.

A selective fluorescence reaction for peptides and chromatographic analysis.

A novel and selective fluorescence reaction is proposed for the quantitative determination of peptides by reversed-phase liquid chromatography (RPLC). A single fluorescent product was formed when a peptide was heated at 120 degrees C for 20 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate, and sodium borate. The fluorescent products of four peptides such as Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, and Leu-Leu-Leu were easily separated on a reversed-phase column by gradient elution of methanol in a mobile phase containing sodium borate (pH 7.0), and then quantitatively detected by fluorimetry. The lower limits (S/N=3) of the detection for the tested peptides were 0.5-1.0 pmol per an injection volume (40 microl). In addition, the fluorescent products of phenylalanine amide and Leu-Leu-Leu were identified by electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS) for the elucidation of their chemical structures.

Facile assay of telomerase activity utilizing a DNA-detectable chemiluminogenic reagent.

Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r2=0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis.

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.

Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker.

We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic acid substrate, followed by fluorescence (FL) detection specific for orotic acid using the 4-trifluoromethyl-benzamidoxime fluorogenic reagent. The DHODH activities in the biologically complex samples were readily measured by the assay method. Our data indicate significantly higher DHODH activity in HeLa cells (340 ± 25.9 pmol/105 cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/105 cells/h), and in malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) than in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). This is the first report that DHODH activity may be a diagnostic biomarker for cancer.

Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe.

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants.

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance.

Dendrimer-like polymeric DNAs as chemiluminescence probes for amplified detection of telomere DNA on a solid-phase membrane.

For the first time, an amplified chemiluminescence (CL) detection of the telomere DNA spotted on a nylon membrane is described here, based on the direct hybridization with the CL probe of dendrimer-like polymeric DNAs possessing a large number of guanine moieties. This probe was synthesized by sense and antisense hybridization between Y-shaped DNAs and then could hybridize with the target DNA.