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P H Corran

Publications and source records attributed to P H Corran.

17 recordsLinked to original sources
PubMed1995

Characterisation of the chains of human fibrinogen isolated by perfusion chromatography using fibrin specific monoclonal antibodies.

In order to study the epitopes on fibrin to which monoclonal antibodies are directed, we required pure individual polypeptide chains of human fibrinogen in milligram quantities. High purity chains of human fibrinogen were rapidly obtained, in under 3 minutes, by the novel procedure of reversed-phase perfusion chromatography and these chains were subjected to immunological characterisation using monoclonal antibodies specific to the individual chains. Cross-reactivity against these antibodies in both immunoblotting and enzyme linked immunospecific assay (ELISA) procedures showed that these isolated fibrinogen chains were of high purity and retained high immunoreactivity. These chains were employed to initiate studies to define the epitopes in fibrin to which four fibrin specific monoclonal antibodies, B10, A11, 5F3 and 1H10 are targeted. Two of these antibodies, B10 and A11, were shown to be directed to a linear sequence on the A alpha chain, although the binding profiles for the two antibodies suggested that different epitopes may be involved for each of these two antibodies. MAbs, 1H10 and 5F3, however, did not bind to any of the three fibrinogen chains, suggesting that conformational epitopes in fibrin are likely to be involved in the binding of these two antibodies to fibrin.

Antibodies, Monoclonal↗
PubMed1994

Ultra-rapid preparation of milligram quantities of the purified polypeptide chains of human fibrinogen.

In order to study the epitopes in fibrin towards which monoclonal antibodies are directed we needed the pure individual polypeptide chains of human fibrinogen in reasonable quantity. We report here a simplified, rapid method of separation of high-purity human fibrinogen chains. Following reduction and S-carboxymethylation of human fibrinogen, the sample was injected directly onto a column of the polymeric reversed-phase perfusion packing POROS 20-R2, and the chains were completely resolved in less than 3 min at a flow-rate of 10 ml/min. The capacity was equivalent to that of a similar sized conventional silica-based column. However the throughput was approximately five to ten times as high. The column was durable and robust in day-to-day use.

Chromatography↗
PubMed1994

Lysine vasopressin undergoes rapid glycation in the presence of reducing sugars.

Lysine vasopressin (LVP) readily reacts with reducing saccharides both in lyophilized preparations and in aqueous solution. Incubation of LVP with, for example, lactose over a pH range of 3.0-8.5 in phosphate buffer or simply in water, gives rise to a number of reaction products, some of which form rapidly (in a matter of hours) even in the frozen state. Reaction mixtures were analysed by reversed-phase HPLC and the structures of the products were deduced from the amino-acid composition of isolated components, by comparison with product profiles obtained with analogues under similar conditions and by FAB mass-spectral analysis of derivatives isolated after reduction with cyanoborohydride. The primary products arise from the formation of Schiff's bases at one or both of the two amino functions. The alpha-amino group of the N-terminal cystine is considerably more reactive than is the epsilon-amino group of lysine and it is the N-terminal adduct which rapidly forms even at -20 degrees C. It is concluded that caution must be shown in using reducing sugars in formulations containing peptides and proteins, particularly the vasopressins and oxytocin.

Amino Acid Sequence↗
PubMed1990

Determination of L-thyroxine in reference serum preparations as the o-phthalaldehyde-N-acetylcysteine derivative by reversed-phase liquid chromatography with electrochemical detection.

A simple procedure for the assay of L-thyroxine in serum preparations with D-thyroxine as internal standard is described. The L-thyroxine is extracted with acetonitrile, fractionated on a reversed-phase silica cartridge and analysed by reversed-phase high-performance liquid chromatography of the o-phthalaldehyde-N-acetyl-L-cysteine derivative. This derivative is not fluorescent, but may be detected with suitable sensitivity and selectivity with an electrochemical detector.

Acetylcysteine↗
PubMed1990

International Standards for salmon calcitonin, eel calcitonin, and the Asu1-7 analogue of eel calcitonin: calibration by international collaborative study.

Regulatory specifications in most countries require that the potency of salmon calcitonin (sCT) clinical products be expressed in International Units (IU) defined by the World Health Organization (WHO) International Standard. The first ampouled standard was prepared in 1972 and has been distributed world-wide since then. A batch of ampoules to serve as the replacement standard is now required. Other piscine calcitonins, eel calcitonin (eCT) and an amino-suberic acid analogue of eCT (Asu1-7 eCT) are now clinical products in some countries and international standards are required for these peptides which are similar to, but not identical with, sCT. This paper describes the preparation of three new ampouled standards and their biological calibration by international collaborative study comprising 17 participants from 10 countries. Following the recommendations in the final report of the collaborative study, the 2nd International Standard (IS) for sCT, the 1st IS for eCT and the 1st IS for Asu1-7 eCT were recently established by WHO, each with an assigned potency in IU, and are now available for issue.

Animals↗
PubMed1982

The International Reference Preparation of Gonadorelin for Bioassay: a comparison with different preparations of synthetic luteinizing hormone releasing hormone using physicochemical methods of analysis, different bioassays and immunoassay.

The preparation and nature of the International Reference Preparation of Gonadorelin for Bioassay (IRP; coded 77/596) are described. The IRP was studied by four laboratories in four countries and compared, using physicochemical methods of analysis, various bioassay procedures and immunoassay, with preparations of synthetic luteinizing hormone releasing hormone (LH-RH) produced by different manufacturers. Analyses by thin-layer chromatography and by reverse-phase high-performance liquid chromatography (HPLC) indicated some heterogeneity of the peptide present in most of these preparations of synthetic LH-RH, including that of the IRP; the latter preparation appeared to be 88.3% (w/w) pure, judged by HPLC. The data from the collaborative study suggested that each ampoule of the IRP contains approximately 31 nmol LH-RH. The IRP appeared to be suitable to serve as an international reference preparation for bioassay since its behaviour was similar in different bioassays, so far as this could be examined, to that of the other preparations of LH-RH with which it was compared. Furthermore, the biological activities of different preparations of LH-RH, assessed in terms of the IRP, appeared to correlate with their degrees of purify assessed by physicochemical methods, suggesting that the peptides other than LH-RH present in the IRP did not contribute significantly to the biological activity of the preparation in these assay procedures. The limited data available suggested that the IRP might also be suitable as a reference preparation for immunoassay. The ampouled preparation, coded 77/596, was therefore established by the World Health Organization as the International Reference Preparation of Gonadorelin for Bioassay and assigned a unitage of 31 i.u./ampoule on the basis that the i.u. is represented by 1 nmole of LH-RH.

Chromatography, High Pressure Liquid↗
PubMed1981

Purification and assay of bovine parathyroid hormone by reversed-phase high performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (HPLC) has been used to purify a crude extract of bovine parathyroid glands, in a single run on an analytical column, to give a high yield of homogeneous material with full bioactivity in in vivo bioassay. Bovine parathyroid hormone (bPTH) prepared and purified by conventional procedures has been rapidly and quantitatively separated from its oxidation and other degradation products, from hormone fragments and from non-hormonal contaminants. Recovery of bPTH, monitored by region-specific immunoassays, in vivo bioassay and re-chromatography on HPLC was greater than 93%. The detection limit of the HPLC system, using endogenous tryptophan fluorescence, was 20 ng bPTH.

Animals↗
PubMed1975

The amino acid sequence of rabbit muscle triose phosphate isomerase.

The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.

Amino Acid Sequence↗
PubMed1975

The reaction of penicillin with proteins.

The mode of reaction of benzylpenicillin with two proteins was studied, with particular reference to the allergenicity of penicillin. These reactions, with pig insulin, and with hen's-egg-white lysozyme, were carried out in neutral solution at 37 degrees C. High concentrations of penicillin are needed to label the proteins, owing to concurrent hydrolysis of penicillin. Evidence has been obtained that the penicillin-reactive sites on the insulin molecule are the alpha-amino group at the N-terminus of the A chain and the epsilon-amino group of the lysine residue; whereas a site of reaction with lysozyme appears to be the epsilon-amino group of lysine-116.

Amino Acids↗
PubMed1974

The tryptic peptides of rabbit muscle triose phosphate isomerase.

1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.

Amino Acid Sequence↗
PubMed1971

Amino acid sequences around the cysteine residue of calf lens -crystallin.

1. Calf lens alpha-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.

Amino Acid Sequence↗
PubMed1969

N-terminal sequences of alpha-crystallin.

The amino acid sequences at the N-terminal ends of the chains of the lens protein, alpha-crystallin, were studied. Both the main kinds of chain in bovine alpha-crystallin (A chains and B chains) have an N-terminal methionine residue, and the amino group is acetylated. Selective purification of the peptides in a tryptic digest of bovine alpha-crystallin gave a preparation consisting largely of the N-terminal peptide from the A chains, and the sequence of this peptide was elucidated. Subsequently, the N-terminal peptides were prepared from separated A and B chains. The proposed sequences are: A chain, acetyl-Met-Asp-Ile-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys; B chain, acetyl-Met-Asp-Ile-Ala-Ile-His-(Pro,Trp)-Ile-Arg. The similarity between the sequences supports the hypothesis that the A and B chains are derived evolutionarily from a common precursor.

Amino Acid Sequence↗