Peter M. Hammond

Development of an enzyme-based assay for acetaminophen

A new and novel method for determination of serum acetaminophen is described. The assay, which can be completed in less than 5 min, is based on the enzymatic hydrolysis of acetaminophen, with subsequent colorimetric detection of the aminophenol so produced. Various possible means of aminophenol estimation are described; the final reaction conditions have been optimized for maximum sensitivity and assay speed. This assay compares favorably with other available procedures; it requires only small sample volumes; it is rapid, simple, and highly specific for the parent drug; and it requires neither great technical ability nor expensive instrumentation.

Development and validation of a robust specific enzyme mediated assay for phenylalanine in serum

The specificity of a phenylalanine dehydrogenase, particularly with respect to cross reactivity toward tyrosine, has been shown to be pH dependent, being minimal at high pH. The dehydrogenase step has been coupled to colorimetric detection of NADH using a tetrazolium salt. The assay shows no significant cross reactivity towards a range of amino acids or drugs and correlates well with an established HPLC technique.

The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.

An enzyme mediated, colorimetric method for the measurement of salicylate.

A novel enzymatic assay for salicylate in serum has been developed. Salicylate monooxygenase and NADH are used to convert the drug to catechol. This is reacted with 4-aminophenol at high pH to yield a blue product, which is detected colorimetrically. The assay is complete in less than seven minutes and requires no sophisticated equipment. The method is precise, sensitive and shows excellent accuracy in recovery experiments and when compared to a high performance liquid chromatography method. The assay is free from interference by coloured or turbid samples, salicylate metabolites, structurally related compounds such as benzoate and 4-hydroxybenzoate, and a range of drugs. The assay reagents demonstrate excellent stability. The formulation of the assay in two stages provides increased specificity and sensitivity compared to other emergency salicylate assays and allows the inclusion of reagents to greatly enhance the stability of the salicylate monooxygenase-NADH reagent, yet the method is simple and performs well.

Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6-1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.

Use of triazine dyes as ligands for the large-scale affinity chromatography of a thermostable glycerokinase

Abstract Glycerokinase from the thermophilic bacterium Bacillus stearothermophilus was found to have an affinity for several reactive, triazine dyes. The enzyme was found to have a strong affinity for several dyes when the dyes were immobilised on an agarose support matrix. Matrices based on cellulose, or polymers of acrylic acid, were less suitable. The study of the effect of various physicochemical properties is described, including ligand concentration, pH, ionic strength, column dimensions and flow-rate. Glycerokinase could be bound to Procion blue MX-3G-Sepharose at low ionic strength (pH 7.5) and was most readily recovered by substrate elution. This matrix was used to purify to homogeneity the enzyme derived from 1 kg of bacteria, in three steps, with an overall recovery of 44%.

Collaborative trial of an enzyme-based assay for the determination of paracetamol in plasma

From the Division of Clinical Chemistry, MRC Clinical Research Centre, Harrow, Middx HAl 3UJ t Department ofClinical Biochemistry, Addenbrookes Hospital, Cambridge CB22QR; ••Poisons Unit, New Cross Hospital, London SE14 5ER; ttDepartment ofClinical Biochemistry, Queen Elizabeth II Hospital, Welwyn Garden City AL7 4HQ; •••Department ofClinical Biochemistry, St Albans City Hospital, St Albans, Herts AL3 5PN;ttt Microbial Technology Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts SP40JG

Recombinant Protein A from Escherichia coli JM83

Protein A is well known for its ability to bind the Fc region of mammalian IgG from a wide range of sources and has been used for the purification of numerous immunoglobulin-based products. It was originally produced from Staphylococcus aureus, although yields were frequently low. The protein has subsequently been purified by a combination of conventional chromatographic techniques or by single-step procedures using affinity matrices. Such affinity matrices, using IgG as a ligand, have traditionally been produced by cyanogen bromide activation, a method that gives rise to some degree of ligand leakage with each column cycle. Furthermore, it is difficult to achieve high ligand levels when using matrices with Row rates compatible with production processes, for example, crosslinked Sepharose. We describe the pilot-scale production of protein A from a high yielding clone as well as the investigation of alternatives to CNBr-activated affinity matrices for downstream processing. A suitable protocol for pilot-scale production of protein A is described.

A Simple Colorimetric Assay To Determine Salicylate Ingestion Utilizing Salicylate Monooxygenase

Salicylate (aspirin) is a common non-steroidal drug, widely used for its analgesic and anti-inflammatory properties. Ingestion of large amounts, however, leads to disturbances of the central nervous system and to gastrointestinal problems, encephalopathy, and renal failure. Salicylate intoxication represents an acute medical emergency; and rapid diagnosis and quantitation of the drug is necessary to assess effective patient management. Salicylate has traditionally been measured by the Trinder reaction, which is based on the interaction between salicylate and ferric ions. There are, however, interference problems.* Other require expensive instrumentation and a high level of technical skill. A further alternative is the enzymic conversion of salicylate to catechol. This can be monitored spectrophotometrically by the concomitant conversion of NAD(P)H to NAD(P): or the catechol can be converted to an indophenol dye,6 and measured colorimetrically. This method is simple to perform and may prove suitable for adaptation to dipstick technology. We report here the large-scale production of salicylate monooxygenase from Pseudomonas cepacia, its purification, and its presentation as a diagnostic reagent in the colorimetric determination of salicylate. Cells were grown in a 500 L vessel, on 2% tryptone soya broth. Sodium salicylate was added as an inducing substrate a t up to 0.4%, during late exponential growth. Cells were grown for about 8 hr a t 37OC, with forced aeration a t up to 600 L/min. The pH was maintained at 6.8-7.2. When the dissolved oxygen fell sharply, the culture was harvested via a chilled plate exchanger using two DeLaval centrifuges running in parallel The culture profile is shown in FIGURE 1. A typical culture yielded 7.5 kg (wet wt) of cell paste, equivalent to 187,000 U enzyme. Frozen bacterial cell paste ( 3 kg) was suspended in 6 L of 0.05 M potassium phosphate buffer, pH 7.0, and allowed to thaw overnight. Cells were disrupted by a single passage through a Manton Gaulin homogenizer a t 55 MPa. Cellulose-based cell debris remover was added; and the preparation was centrifuged at 5,000 x g for 8 hr. The cell-free extract was purified by chromatography on a DEAE-Sepharose ionexchange column ( FIG. 2). This was followed by hydrophobic interaction chromatography on phenyl Sepharose, with elution using 0.01 M Tris/HCl, pH 9.0, containing 10%

Large-scale purification of the chromosomal β-lactamase from Enterobacter cloacae P999

Abstract Homogeneous β-lactamase (β-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the β-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.