Spectrophotometric Assay of Endo-β-N-acetylglucosaminidase H Activity

Abstract

An endoglycosidase H (Endo H) assay based on the use of a glycoprotein enzyme as substrate, in conjunction with immobilised concanavalin A (Con A), was devised and investigated.

In this system, ribonuclease B (RNAase B) functions as both glycosidase substrate and reporter enzyme. Separation of glycosylated from deglycosylated RNAase B is effected by mixing with colloidal Con A-agarose, followed by centrifugation. The activity of carbohydrate-denuded RNAase B in the supernate is used to calculate endoglycosidase activity.

Results obtained with this assay correlated closely with analysis of digest products by denaturing electrophoresis and lectin reactivity on western blots. Effective removal of non-hydrolysed RNAase B from assay digests was dependent on both lectin-agarose bead concentration and time of exposure.

The assay was linear over the range 10–100 fmol of Endo H protein; a K_m value of 0.48 mmol/l and turnover number of 7200 mol RNAase B/mol enzyme/min was determined for this endoglycosidase.

The suitability of the assay for measuring the activity of other endoglycosidases was also investigated, and the usefulness of the assay is discussed.     

A Microtiter Plate Assay for Sulfonamides

Abstract

Sulfamethoxazole, sulfisoxazole, and sulfadiazine are sulfonamides used in the treatment of several infectious diseases. Several studies have demonstrated that the amino substituent plays an important role in both the toxicity and the therapeutic effect of these drugs. In view of these findings, a rapid and convenient method of analysis would be useful for monitoring selected patients receiving these drugs. With the increasing use of microtiter plate methodology in the clinical laboratory, an assay based upon the Bratton-Marshall reaction with the amino substituent was adapted to the microtiter plate format. The results indicate that the microtiter plate assay for sulfonamides retains the sensitivity and linearity necessary for analysis of sulfonamides in biological fluids at clinically relevant concentrations. The assay is simple, rapid, convenient, and suitable for monitoring procedures where only the measurement of the active drug concentration is required.     

Improved Fluorescence Assay of Liposome Lysis

Abstract

A simple fluorescence assay for liposome stability is described. A fluorogenic enzyme substrate. 4-methylumbelliferyl-β-D-glucuronide, is encapsulated in liposomes. In the presence of E. Coli β-glucuronidase, any of the substrate that has been released into the medium is converted to 4-methylum-belliferone, which is monitored fluorimetrically. The method offers several advantages over current methods, including high gain, extremely low leakage, and facile quantitation. The use of the assay as part of a prototype immunoassay is decribed.     

Fluorescence Assay for Membrane Lytic Peptides

Abstract

Lytic peptides such as melittin and mastoparan are usually assayed by measuring the leakage of cell contents; e.g., hemolysis. When such peptides lyse liposomes containing concentration-quenched 6-carboxyfluorescein (6CF), the resulting fluorescence increase is proportional to the amount of lytic peptide added. Using this 6CF-liposome system, one can assay nanogram quantities of melittin. A protocol was developed to survey peptides for lytic activity and at the same time, to test for mast cell degranulating activity. Peptides possessed either, both, or neither of these activities. The dye-liposome system was used to assay HPLC fractions of bee venom. This fluorescence assay for lytic activity is more sensitive and convenient than the hemolysis method, does not require removal of unlysed structures, and does not require animal cells.     

A Spectrophotometry Assay of Phosphodiesterases

Abstract

A very sensitive analytical technique is described for measuring phosphodiesterase (PDE) activity in mouse muscle, where levels of these enzymes are very low. After the action of PDE, a second enzyme, 5′nucleotidase, is employed to release inorganic phosphorus (Pi) which is assayed by a colorimetric method using malachite-green as dye solution. It is thus possible to measure very small amounts of Pi (0.1 – 0.5 μg) and to assay PDE activity with all the cyclic nucleotides even in the presence of methyl-xanthines. Phosphodiesterase assay in this manner gives a response that is linear with serial amounts of purified enzyme and different quantities of muscle homogenate.     

Enzyme-Amplified Receptor Assay Predictive Model and Analytical Limits

Abstract

A theoretical basis for enzyme amplified receptor assay (ERA) is presented and criteria are established for the general case of any receptor system. Analytical criteria necessary to perform this assay are identified based upon the relevant affinity constants, the concentrations of the enzyme-labelled ligand and receptor, and the analyte.     

HPLC Assay of Phospholipase A2 Activity Using Low-Temperature Derivatization of Fatty Acids

Abstract

A modified isocratic reverse phase high-performance liquid-chromatographic (RP-HPLC) method for phospholipase (PLA) activity assay is developed. Natural lecithins and synthetic phospholipids are used as substrates, and released fatty acids are analyzed after single-phase derivatization (with p-bromophenacyl bromide) at low temperature. The procedure allows simultaneous determination of the total and specific phospholipase activity. This method was successfully applied to snake venom PLA₂ activity assay using natural (soybean and egg yolk lecithins) and synthetic (dipalmytoylphosphatydylcholine) substrates for quantitative determination of the enzyme activity.     

Enzyme-Amplified Receptor Assay (ERA): A Novel Approach to Drug Detection

Abstract

Isolated acetylcholine receptors from Torpedo nobiliana are used as recognition elements for phencyclidine (PCP), a potent hallucinogen with high affinity for the ion channel site associated with the receptor, in a novel enzyme-amplified receptor assay. A fixed amount of PCP labelled enzyme competes with free PCP for a limited number of receptor sites resulting in changes of observed enzyme activity proportional to free drug concentration. The proposed concept combines the use of receptors with an enzyme amplification step to yield an exceptionally simple and convenient assay.     

Testosterone Determination Using Rapid Heterogeneous Competitive-Binding for Enzyme-Linked Immunosorbent Assay in Flow Injection.

Abstract

The application of a rapid competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of testosterone in flow injection (FI) is presented. The ELISA protocol for the determination of testosterone was performed in 60 min compared to the standard protocol time of 150 min. This was achieved by reducing the antibody incubation time. The ELISA was used successfully to determine testosterone levels in clinical samples of both male and female subjects, and were in good agreement compared to a pathology laboratory which employed an immunoradiometric assay (IRMA).     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

An Automated Spectrophotometric Flow Injection Assay of Alkaline Phosphatase

Abstract

Assay of alkaline phosphatase using an automated flow injection manifold is described. The detector used is a spectrophotometer. The measurement is based on the rate of formation of p-nitrophenol at 405 nm, from the substrate p-nitropheny1 phosphate. The linear range, precision and accuracy of the technique for the assay of the enzyme is given. The advantages of the approach to assay of plasma enzymes are also discussed.     

Comparison of Different Assay Methods for Determination of Elastase Activity

Abstract

Four different methods of pancreatic elastase (E. C. 3.4.21.11) assay have been compared: one use N-succinyl-(Ala)₃-paranitroanilide as a substrate; the three others use elastin, modified or not.

The former has been monitored either with a spectrophotometric or a conductimetric device.

All methods give a linear relationship with amounts of pancreatic elastase in the range 0–5 μg.

The conductimetric method is the only one using unmodified elastin which measures elastolysis in initial rate conditions, avoiding artificial amplification of hydrolysis if other proteases are present.     

Assay of some Clinically Important Reductants by a Chemiluminescence-Delay Technique

Abstract

A chemiluminescence-delay technique has been developed for the assay of clinically important reductants. Under the experimental conditions described the time de lay before chemiluminescence is observed exhibits a linear dependence on substrate concentration up to ～ 6 × 10^?5 M. The method, utilises the ferrihaem-catalysed oxidation of luminol by hydrogen peroxide as the light-emitting reaction. Specificity is conferred upon the assay by the use of the appropriate oxidoreductase enzymes. The technique has been applied in the estimation of ascorbic acid and uric acid in aqueous standards and biological samples.     

A Simple,But Reliable Assay of Sex Hormone Binding Globulin

Abstract

The likelihood of erroneous results is very high when a single dose of ligand (e.g. dihydrotestosterone) is employed in the assay of sex hormone binding globulin (SHBG) using precipitation with ammonium sulfate. However, correct results will be obtained if several ligand doses are used and the calculations are based on a Scatchard plot from which non-specific binding has been eliminated. The reliability of such a multiple-dose SHBG assay was tested. As established by an analysis of variance, the results of the measurements were independent of the volume assayed. Furthermore, by assaying 25 plasma sample in duplicate, an average within-assay coefficient of variation of 5.5% was obtained. The assay of the same samples on different occasions gave an estimate of between-assay variation ranging from 3.5% to 8.9% for various types of plasma. Moreover, the results of the assay of 170 plasma samples were well correlated (r = 0.8) with those obtained by a steady state electrophoresis. Thus the multiple-dose precipitation assay gives reliable results and is suitable for routine measurements of SHBG.     

Assay of N-Propylajmaline in Blood Plasma by Ion-Pair Liquid-Liquid Chromatography

Abstract

A sensitive method for assay of N-propylajmaline (prajmaline) in human plasma is described. The quaternary ammonium compound exists as a pair of stereoisomers, which are isolated and separated by ion-pair liquid-liquid chromatography on microporous silica particles. An aqueous solution containing perchloric acid and sodium perchlorate is used as stationary phase and a mixture of butanol, dichloroethane and hexane as mobile phase. The procedure involves ion-pair extraction from plasma and evaporation prior to the chromatographic separation. Selective detection is achieved by using a fluorescence detector. The method allows assay of concentrations down to 10 pmol of the two forms of prajmaline in 1 ml of plasma with a relative standard deviation below 5 %.     

The Simultaneous Spectrophotometric Assay of the Active Constituents of Multicomponent Analgesics Using Kalmanfiltering

Abstract

UV-spectrophotometry in combination with Kalmanfiltering is used for the assay of analgesics in tablets. Commercial products containing various combinations of acetylsalicylic acid, salicylic acid, caffeine, phenacetine and acetaminophen are analyzed. For comparison, these tablets were also analyzed by HPLC. The accuracy and precision of both methods are comparable. The described spectrophotometric assay with the aid of Kalmanfiltering represents a new, rapid and low-cost alternative to existing analytical procedures for analgesics.     

Further Studies on the Interaction of Immobilized Folate Binding Protein and Enzyme-Folate Conjugates; An Enzyme-Linked Competitive Binding Assay for Folate

Abstract

The kinetic and thermodynamic properties of the folate interaction with immobilized folate binding protein (FBP) are examined. The use of enzyme-rather than radio-labeled folate provides insight into the complex binding mechanism of folate with FBP and indicates that polymerization of the binding protein (evident for FBP-folate association in solution) is not a prerequisite for the cooperative behavior observed. An enzyme-linked competitive binding assay for folate based on this interaction is described and dose-response curves demonstrate the sensitivity and selectivity of the method. The accuracy of the assay is tested by determining folate in infant formula.     

Determination of Propylthiouracil in Human Breast Milk by Direct Injection High Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic(HPLC) method was developed for the assay of propylthiouracil in human breast milk. After filtration with membran filter(Molcut II), the eluent was injected into a liquid chromatogaph equipped with C₁₈ precolumn and analytical column in series according to column switching techniques. This method is sufficiently sensitive for most pharmacokinetic studies in human breast milk. The concentration of propylthiouracil was linear over the 50 – 5000ng/ml range. The recovery and the coefficient of variation was 92.0 – 100.6% and 1.6 – 2.9%, respectivery. This assay has the advantages of specificity, simplicity and reproducibility for the measurement of propylthiouracil in human breast milk.     

Determination of Toxins Using Enzyme-Amplified Receptor Assay

Abstract

A new receptor based assay is described for the determination of toxins which have high affinities for the acetylcholine receptor. The method is based upon the hindrance of the normal binding of a synthetic enzyme-drug conjugate with a high affinity for the acetylcholine receptor protein by the presence of toxins acting as antagonists. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate for NADH formation at 340 nm. The procedure proposed is designed to provide a simple toxin screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for snake venoms from Bungarus multicintus, Naja naja, and the alkaloid tubocurarine. Aspecific binding responses are shown to have minimal effect on the assay.     

Fluorimetric Assay for Serum Albumin Based on Its Enzymatic Activity

ABSTRACT

Serum albumins are known to catalyze the hydrolysis of aryl esters. Both human and bovine albumins are active against Naphthol AS acetate, resulting in a fluorescence excited at 320 nm and monitored at 500 nm. HSA was more active than BSA. At pH 8.0 the reaction is activated by cetyltrimethylammonium bromide. Other esterases in serum require either calcium or a higher pH for activity. The assay is conducted with albumin diluted to about 10^?7 M or less, thus dissociating many potentially interfering ligands. Palmitic acid did not interfere. The interference by bilirubin is minimized by using highly dilute albumin. The present method gives results with serum which correlate well with the widely used Bromcresol Green method. Limit of detection for HSA is 14 picomoles.