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Biomedical subjects

H B Halsall

Publications and source records attributed to H B Halsall.

At least 55 records · Page 3Linked to original sources
PubMed1985

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.

Acrylamide↗
PubMed1985

Collagen fibril formation in the presence of sodium dodecyl sulphate.

Sodium dodecyl sulphate (SDS) was used to weaken both the electrostatic and the hydrophobic interactions during collagen fibrillogenesis in vitro. The rate and extent of fibril formation as well as fibril morphology were affected by SDS concentration. Both the formation of large fibrils at 0.3 mM-SDS and the complete cessation of fibril formation at 0.5 mM-SDS were considered to be the result of SDS-induced conformational changes in the non-helical telopeptides. A possible mechanism of SDS interaction with the N-terminal and the distal region of the C-terminal telopeptides is offered.

Collagen↗
PubMed1985

Redox activation of galactose oxidase: thin-layer electrochemical study.

The redox activation of galactose oxidase by various oxidants is characterized by using a unique thin-layer electrochemical cell. Activation is shown to be strictly a redox process and can be rapidly accomplished by using ferricyanide, cobalt terpyridine or tetracyanomonophenanthroline ferrate, and a control electrode to control solution potential. This oxidation is a one-electron process and does not result in modified galactose oxidase which exhibits enhanced activity. On the contrary, this oxidation is required for activity. The solution potential dependence of activity is independent of which of these mediator-titrants is used, the concentration used, and which of various substrates is used in the determination. The substrates used were acetol, dihydroxyacetone, glycerin, 2-propyn-1-ol, allyl alcohol, 2-butyne-1,4-diol, furfuryl alcohol, benzyl alcohol, 4-pyridylcarbinol, galactose, and stachyose. The E1/2 and n values obtained are 0.40 +/- 0.005 V vs. SHE and 0.9 +/- 0.1 electron at pH 7.3. E1/2 is defined as the potential at which half the maximal enzymatic activity is observed and probably reflects the E0' of the enzymic group involved in activation. A model is proposed in which activation occurs during turnover due to the redox buffering (by oxidants) of an enzymic Cu(II)/Cu(I) state which has a higher E0' than in resting galactose oxidase. The pH dependence of E1/2 is 60 mV/pH unit in the pH range 6.0-8.0. The data suggest that the deprotonation of an amino acid residue facilitates the one-electron oxidation (activation) of galactose oxidase.

Electrochemistry↗
PubMed1985

Heterogeneous enzyme immunoassay with electrochemical detection: competitive and "sandwich"-type immunoassays.

In these competitive and "sandwich"-type heterogeneous enzyme immunoassays, based on liquid chromatography with electrochemical detection, rabbit immunoglobulin G is used as a model compound. Alkaline phosphatase (EC 3.1.3.1), the labeling enzyme, catalyzes conversion of phenyl phosphate to phenol. After separation on an octyldecylsilane column, the enzyme-generated phenol is detected in a thin-layer cell at a carbon-paste working electrode. The detection limit for phenol is 5.0 nmol/L. The electrode response varies linearly with concentration over a range of three orders of magnitude. For the sandwich-type assay procedure the detection limit is 10 ng/L; the linearity ranges over four orders of magnitude. The detection limit of the competitive immunoassay is 5 micrograms/L. The dynamic range spans two orders of magnitude.

Alkaline Phosphatase↗
PubMed1984

Crystallization of alpha 1-acid glycoprotein.

Crystals of alpha 1-acid glycoprotein have been grown reproducibly from delipidated protein in the presence of chlorpromazine. The crystals are large hexagonal prisms of space group either P622 or P6(2)22 and the unit cell dimensions are a = b = 101 A and C = 201 A. The unit cell is very highly hydrated and is nearly 80% solvent. It contains one molecule of protein per asymmetric unit. The crystals diffract only to low resolution, presumably reflecting the extensive hydration and accompanying disorder.

Chlorpromazine↗
PubMed1984

A polycationic amine that induces unique conformational changes in poly(dA-dT) in low salt.

Circular dichroism was used to examine alterations in the secondary structure of poly(dA-dT) X poly(dA-dT) upon binding polymer X, a polycationic CD probe for aspects of DNA structure. Stable complex formation is evidenced by increasing Tm and the appearance of large extrinsic bands in the greater than 300 nm, region which increase proportionally with r (ratio of polymer charge to DNa phosphate), in the range 0.0 to 0.32. At relatively low values of r (less than .32), CD spectra of the poly(dA-dT) X poly(dA-dT)-polymer X complex show a gradual non-cooperative inversion in the long wavelength portion (275 nm) of the intrinsic band in low salt solutions suggesting structural and conformational flexibility in poly(dA-dT) X poly(dA-dT) and further implicating polymer X as a potential probe for variations in DNA secondary structure. The dinucleotide repeat configuration of poly(dA-dT) X poly(dA-dT) is presumed to play a role in the observed intrinsic CD changes. NMR data support an "alternating B" conformation for the complex.

Circular Dichroism↗
PubMed1983

Metal complexes as mediator-titrants for electrochemical studies of biological systems.

Various redox-active metal complexes were evaluated electrochemically for stability, reversibility, and general utility as mediator-titrants in the 0 to + 1.0 V vs standard hydrogen electrode range. Those complexes which were evaluated and appear to be useful in potentiostatic or potentiometric techniques are Ru(bpy)3(CIO4)2, Ru(bpy)2(im)2, Os(bpy)3(NO3)2, K2Fe(phen)(CN)4, Co(phen)3Cl2, and Co(terpy)2Cl2.

Chemical Phenomena↗
PubMed1983

Synthesis of photoaffinity analogs of progesterone.

A photoaffinity analog of progesterone, 11 alpha-diazoacetate progesterone, has been synthesized by a two step procedure. This has been made possible by a single reaction combining both esterification and diazotization. Numerous diazoacetate analogs of progesterone can be synthesized using these simple reaction conditions. Radioactive analogs can be produced that transfer the isotope covalently to the site of protein modification upon photolysis.

Affinity Labels↗
PubMed1982

The binding of spin-labeled propranolol and spin-labeled progesterone by orosomucoid.

The binding of the spin-labeled propranolol and spin-labeled progesterone to human orosomucoid has been studied as a function of temperature by electron spin resonance (ESR) techniques. At 20 degrees C the association constants are 1.9 x 10(6) and 4.9 x 10(5) M-1, respectively. In each case, the binding is competitive with unlabeled ligand. Above about 50 degrees C the apparent association constant for both ligands decreases rapidly with increasing temperature. This is due to thermal denaturation of the orosomucoid, as was shown independently by ultraviolet absorption spectroscopy and differential scanning calorimetry. Below the denaturation region the number of binding sites per orosomucoid molecule remains constant at approx. 1. Examination of the thermodynamic parameters shows the progesterone binding at 37 degrees C to be essentially enthalpically driven, while the propranolol binding at 37 degrees C has a substantial entropic component.

Carrier Proteins↗
PubMed1982

The preparation of orosomucoid from nephrotic urine.

A gel chromatographic and ion exchange procedure is described for the preparation of orosomucoid from nephrotic urine. The removal of endogenous lipid and glycosaminoglycan than yields a highly homogeneous preparation. Aggregates are formed at ambient temperatures and as a result of lyophilisation of ultrafiltration.

Freeze Drying↗
PubMed1982

Enzyme immunoassay with flow-amperometric detection of NADH.

Using phenytoin as a model analyte, we demonstrate that an enzyme-coupled immunoassay based on flow-injection analysis and amperometric detection of NADH is both feasible and practical. Good agreement with a routine clinical laboratory procedure for phenytoin was obtained for patients' serum samples. The electrode must be protected to prevent fouling by proteins in the analytical sample. The optimum detection range for NADH was at 0.01 of the concentrations of NADH generated during the several minutes required for each analysis. This suggests that the electrochemical technique should be extendable to the determination of species at concentrations in the microgram per liter range.

Electrochemistry↗