A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase

A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched β-cyclodextrin (Glc_n-β-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (K_m = 0.66 ± 0.02 mM and k_cat = 76.7 ± 1.5 s⁻¹) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis.     

Enhancement of the chiroptical response of α-amino acids via N-substitution for molecular structure determination using vibrational circular dichroism

The chiroptical response in the form of vibrational circular dichroism (VCD) in the midinfrared region is found to be enhanced when a hydrogen of amino group of l -tryptophan is substituted with acetyl, acryloyl, or maleyl group. The order of preference for VCD enhancement is found to be acryloyl > acetyl > maleyl group. The resulting experimental VCD spectra are also found to be satisfactorily reproduced by the quantum mechanical (QM) predicted spectra. The QM predicted spectra were simulated using the conformer populations, (a) predicted by Gibbs energies and (b) optimized to maximize the similarity between experimental and predicted VCD spectra. It is found that the conformer populations predicted by Gibbs energies do not yield the maximum possible similarity between experimental and the QM predicted spectra. This work identifies the N-substitution of α-amino acids and determining the conformer populations that best reproduce the experimental spectra as two new approaches for molecular structure determination.     

A continuous spectrophotometric assay for pyrimidine-5′-nucleotidase

A simple method is presented for the determination of pyrimidine-5′-nucleotidase activity using a continuous spectrophotometric assay system. Activity is determined by measuring inorganic phosphate generation using a linked indicator system that produces uric acid in the presence of inosine, purine nucleoside phosphorylase, and xanthine oxidase. This method has several advantages over any of the methods currently in use.     

Potent dialkyl substrate-product analogue inhibitors and inactivators of α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis by rational design

Rational approaches for the design of enzyme inhibitors furnish powerful strategies for developing pharmaceutical agents and tools for probing biological mechanisms. A new strategy for the development of gem-disubstituted substrate-product analogues as inhibitors of racemases and epimerases is elaborated using α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis (MtMCR) as a model enzyme. MtMCR catalyzes the epimerization at C2 of acyl-CoA substrates, a key step in the metabolism of branched-chain fatty acids. Moreover, the human enzyme is a potential target for the development of therapeutic agents directed against prostate cancer. We show that rationally designed, N,N-dialkylcarbamoyl-CoA substrate-product analogues inactivate MtMCR. Binding greatly exceeds that of the substrate, (S)-ibuprofenoyl-CoA, up to ∼250-fold and is proportional to the alkyl chain length (4–12 carbons) with the N,N-didecyl and N,N-didodecyl species having competitive inhibition constants with values of 1.9 ± 0.2 μM and 0.42 ± 0.04 μM, respectively. The presence of two decyl chains enhanced binding over a single decyl chain by ∼204-fold. Overall, the results reveal that gem-disubstituted substrate-product analogues can yield extremely potent inhibitors of an epimerase with a capacious active site.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

A radioenzymatic assay for femtomole determination of catecholamines using α-methyldopamine as an internal standard

A radioenzymatic assay has been developed for the sensitive determination of plasma catecholamines in perchloric acid extracts using α-methyldopamine as an internal standard. With 25 μl of plasma extract in a total volume of 40 μl the assay gives blank values equivalent to approcximately 2 femtomoles (fmole) for epinephrine (E), norepinephrine (NE), 6 fmole for α-methyldopamine (MeDA) and approximately 15 femtomoles for dopamine (DA). Recoveries of 25 dpm/fmole NE, 40 dpm/fmole E, 56 dpm/fmole DA and 80 dpm/fmole MeDA have been obtained. The assay is linear to at least 1 picomole catecholamine (CA) and shows less than 0.5% crossover between E, NE and DA and a 4.7% crossover of αMeDA into DA. The interassay variability was ± 7% for DA, ± 4% for E and ±3% for NE.     

A continuous spectrophotometric assay for cyclic 3′,5′-nucleotide phosphodiesterase

In the course of studies on cyclic nucleotide phosphodiesterase, the need for an assay which was both sensitive and continuous was realized. Most of the methods available for monitoring phosphodiesterase either depend on the use of accessory enzymes, and are accordingly subject to intrinsic limitations, or are not capable of continuously monitoring the enzymatic reaction. The present paper describes a new spectrophotometric assay for cyclic nucleotide phosphotidesterase which is highly reproducible, rapid, simple, and more sensitive than many of the previously published assays for this enzyme. The method is sensitive enough to detect the enzymatic conversion of 75 pmol of cAMP to 5′-AMP per minute. This assay is based on the fact that the hydrolysis of cyclic nucleotides to the corresponding 5′-phosphate ester by phosphodiesterase makes available an additional titratable proton of the 5′-phosphate group. By incorporating phenol red into the assay mixture, the rate of proton production can be continuously measured by monitoring the decrease in absorbance of the basic chromophore of phenol red at 560 nm. The primary advantages of the spectrophotometric assay described here are: (a) it provides initial velocity measurements, and thus is ideally suited to studying the kinetic properties of partially purified preparations of enzyme, and (b) it does not require the tedious and time-consuming purification of commercially available substrates which is often required when radioisotopic assays are used in detailed kinetic studies. The chief limitations are: (a) the sensitivity is not sufficient to accurately monitor the “low K_m” enzyme, and (b) the use of the assay to quantitate revoveries throughout extensive purification, where different buffer salts at different pH values are used, would require that the enzyme be dialyzed prior to assay.     

Vibrational circular dichroism of tetraphenylporphyrin in peptide complexes? A computational study

The Raman and absorption spectra of tetraphenylporphyrin (TPP) were calculated and compared to experiment. The computation was based on the harmonic molecular force field and electric tensors obtained ab initio at the BPW91/6-31G* level. Good agreement was found between experimental and calculated frequencies and intensities. In order to estimate whether induced optical activity in chiral complexes interferes with the signal of peptide vibrations, the vibrational circular dichroism (VCD) spectra of TPP were simulated. The magnetic field perturbation theory (MFP) and the gauge-invariant atomic orbitals (GIAO) were used for the simulation. Such spectra were compared to theoretical VCD intensities of a model tripeptide as well to experimental spectra of a complex of the peptide and tetrakis(p-sulfonatophenyl)porphyrin (TSPP). No significant contribution to VCD signal from the TPP residue was found in experimental spectra. Thus, possible peptide conformational changes occurring during the complexation can be monitored directly in the amide I frequency region.     

A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.     

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.     

Continuous production of α-peptide using immobilized recombinant yeast cells: A model for continuous production of foreign peptide by recombinant yeast

α-Peptide, a portion of Escherichia coli β-galactosidase, was cloned downstream of the yeast α-factor promoter and the signal peptide by one of the authors. In this study, we utilized recombinant yeast cells, transformed the α-peptide secretion vector and attempted continuous production of α-peptide as a model of foreign peptide production. The continuous production of α-peptide was performed by using immobilized recombinant yeast cells on a column reactor, after characterizing the secretion, using minimal and complex medium. Utilizing minimal medium, with a productivity of 100 000 U h⁻¹ l⁻¹, α-peptide was continuously produced for more than 200 h. We then attempted to improve the productivity of α-peptide by alternating minimal and complex medium. Utilizing this medium changing method, 1.4 times higher α-peptide was produced during 150 h of operation compared with that achieved only by feeding minimal medium.     

Two-site assay of human α1-fetoprotein using125I-labelled monoclonal antibodies

Monoclonal antibodies to human ₁-ietoprotein (AFP) have been compared with a conventionally produced antiserum using radioimmunoassay and two-site immunoradiometric techniques. A low-affinity antibody, which proved inadequate for use in a radioimmunoassay, gave asatisfactory dose-response Curve in a rapid two-site assay. A higher-affinity antibody yielded a simple, rapid, and sensitive two-site assay suitable for routine measurement of serum AFP.     

A dynamic membrane reactor with immobilized α-chymotrypsin for continuous kyotorphin synthesis in organic media

-Chymotrypsin was covalently attached to an -alumina ultrafiltration membrane coated with an inert protein. This derivative was used as catalyst for the continuous kinetically controlled synthesis of kyotorphin in both membrane and packed bed reactors, using aqueous (water/dimethyl sulfoxide, 60:40, v/v) and nearly-dry (hexane/ethanol/water, 57:40:3, by vol.) organic media, respectively. In both media, the synthetic activity and operational stability of the enzyme-membrane derivative was compared with an adsorbed -chymotrypsin Celite derivative, being 2-times and 4-times respectively, higher than the Celite one. The enzyme-membrane derivative showed half-life times higher than 36 days, with a selectivity near to 100%.     

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase

γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.     

A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction

Increasing evidence indicates that copy number variants (CNVs) have great relevance to common human diseases. In α-thalassemia, clinical phenotypes are related to genotypes, specifically copy number changes in the human α-globin gene cluster. Assays are available for high-throughput screening of unknown CNVs genome-wide and also for targeted CNV genotyping at loci associated with genetic disorders. Here we describe a universal quantitative approach based on nested real-time quantitative polymerase chain reaction for accurate determination of copy numbers at multiple particular gene loci. We used the α-globin gene as a model system, obtaining the reproducibility and sensitivity to analyze different gene copies and testing 95 DNA samples with 16 different known genotypes. Our results showed that this approach has high sensitivity and low standard deviations for correctly genotyping DNA samples containing different copy numbers of the α1 and α2 globin genes. Our method is rapid, simple, and reliable, and it could be used to simultaneously screen for α-thalassemia deletions or triplications. Moreover, it has potential as a versatile technology for the rapid genotyping of known CNVs in a targeted region.     

A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine γ-lyase catalysis

In this article, we present a new, easy-to-implement assay for methionine γ-lyase (MGL)-catalyzed γ-elimination reactions of l-methionine and its analogues that produce α-ketobutyrate (α-KB) as product. The assay employs ultraviolet–visible (UV–Vis) spectrophotometry to continuously monitor the rate of formation of α-KB by its absorbance at 315 nm. We also employ a nonlinear data analysis method that obviates the need for an “initial slope” determination, which can introduce errors when the progress curves are nonlinear. The spectrophotometric assay is validated through product analysis by ¹H NMR (nuclear magnetic resonance), which showed that under the conditions of study l-methionine (l-met) and l-methionine sulfone (l-met sulfone) substrates were converted to α-KB product with greater than 99% yield. Using this assay method, we determined for the first time the Michaelis–Menten parameters for a recombinant form of MGL from Porphyromonas gingivalis, obtaining respective k_cat and K_m values of 328 ± 8 min⁻¹ and 1.2 ± 0.1 mM for l-met γ-elimination and 2048 ± 59 min⁻¹ and 38 ± 2 mM for l-met sulfone γ-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL γ-elimination reactions that produce α-KB as the end product.     

A sandwich-type enzyme-linked immunosorbent assay for the analysis of recombinant human interferon α-2b

The ability of a previously developed sandwich-type enzyme-linked immunosorbent assay (ELISA) for discriminating incorrectly folded recombinant human interferon -2b (IFN-2b) molecular species from multi disulphide-bonded species was investigated. This ELISA was applied to evaluate and improve the effectiveness of the renaturation of IFN-2b, a step that is currently used in the large-scale production of IFN-2b produced in recombinant Escherichia coli strains.     

Stereoselection in the binding of Ln DOTA to γ-cyclodextrin: A near infrared circular dichroism study

The formation of an inclusion complex between γ-cyclodextrin and Ln DOTA has been reported in the literature. All Ln DOTA complexes in solution give rise to a network of equilibria between different stereoisomers, which are pairs of enantiomers. By means of near infrared circular dichroism of the Yb derivative, we demonstrate that upon the formation of the host-guest complex, there is a complete stereoselection and that only the Λ(δδδδ) binds.