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P M Hammond

Publications and source records attributed to P M Hammond.

At least 19 recordsLinked to original sources
PubMed1992

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.

Amino Acid Oxidoreductases↗
PubMed1992

The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.

Aspirin↗
PubMed1992

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.

Ammonia-Lyases↗
PubMed1992

Production and purification of salicylate monooxygenase from Pseudomonas cepacia ATCC 29351.

Salicylate monooxygenase (EC: 1.14.13.1) has been produced and purified from Pseudomonas cepacia ATCC 29351 which has the ability to utilise salicylate as a sole carbon source. The bacterium was grown on a defined medium containing 2% (w/v) casamino acids and 0.15% (w/v) yeast extract at 25 degrees C; salicylate monooxygenase production was induced by the presence of up to 0.7% (w/v) sodium salicylate, to a level of approximately 2% of the soluble cell protein. The enzyme was purified over 50-fold, with a recovery of about 40%, by a combination of ion exchange and hydrophobic interaction chromatography. The purified enzyme had a specific activity of 14-15 U mg-1 protein and was essentially homogeneous.

Bacterial Proteins↗
PubMed1990

Development and validation of an automated enzyme assay for paracetamol (acetaminophen).

A rapid, enzymatic assay for serum or plasma paracetamol has been developed with the potential for adaptation to a wide range of clinical analysers. The method involves the action of an amidase enzyme to produce 4-aminophenol from paracetamol, which in turn reacts with 8-hydroxyquinoline in the presence of manganese ions to form a blue dye. Two stable reagents are used and excellent precision is achieved over the drug concentration range 0-2.5 mmol/l. The method, which is complete within 6 min, has been validated using a Monarch centrifugal analyser and shows no significant interference from endogenous serum compounds, drugs or paracetamol metabolites.

Acetaminophen↗
PubMed1990

Development and validation of an automated, enzyme-mediated colorimetric assay of salicylate in serum.

This salicylate-specific assay can be adapted for use with most discrete analyzers, for rapid emergency or routine testing with small serum or plasma sample volumes and a single calibration. The basis of this method is as follows: salicylate monooxygenase (EC 1.14.13.1) converts salicylate to catechol in the presence of NADH; the catechol then reacts with 4-aminophenazone under alkaline conditions, catalyzed by manganese ions, to produce a red dye. Incorporation of an NADH-regenerating system, involving glucose and glucose dehydrogenase, into the enzyme reagent ensures that the working reagent is stable for more than two weeks. The standard curve is linear over the drug concentration range 0 to 5 mmol/L. The CV was less than 4% over 20 days. Results correlated well with those by the Trinder colorimetric method and an HPLC method. We saw no interference by any of 80 drugs we tested at therapeutic concentrations or by endogenous compounds in serum.

Autoanalysis↗
PubMed1988

Large-scale purification of the chromosomal beta-lactamase from Enterobacter cloacae P99.

Homogeneous beta-lactamase (beta-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 beta-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.

Anti-Bacterial Agents↗
PubMed1988

A rapid, enzymatic method for the determination of chloramphenicol in serum.

A simple, rapid assay of serum chloramphenicol has been developed which combines the specificity of the enzyme chloramphenicol acetyltransferase with the convenience of a colorimetric detection system. The assay is linear over the drug concentration range 5-200 microM (1.5-65 mg/l) and therefore is suitable for detection below and above the therapeutic range (31-62 microM, 10-20 mg/l with potential toxicity above 75 microM, 24 mg/l). This method does not detect the microbiologically inactive succinate or palmitate pro-drugs of chloramphenicol and evidence suggests that the major metabolite, chloramphenicol glucuronide also is not detected. Good correlation with an HPLC method has been achieved (r = 0.9860). The assay is based on a two reagent system with very simple methodology, the only instrumentation required being a spectrophotometer. However, the assay could be adapted to run on a range of discrete analysers.

Bilirubin↗
PubMed1986

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.

Buffers↗