A quantitative gel-filtration method for analysis of the proteinaceous fraction of Cheddar cheese

SummaryMethods are described for the solubilization of the nitrogenous portion of cheese in a dissociating solvent system and separation of the solution into fractions on a Sephadex G-100 column under conditions where the proteins and peptides would be expected to be split into monomeric units. TheE280measurement of the fractions, expressed as a percentage of the total material eluted, followed a reproducible pattern, both with duplicate runs on the same sample and comparative runs on non-identical samples of the same type. With cheese curd, a small peak of undissociated material was obtained at the void volume of the column; this was followed by a main peak and a minor third peak, with a small quantity of material being eluted at larger volumes. A qualitatively similar pattern was obtained with 43-day-old cheese as the sample, but the main peak was smaller, the third peak was larger, and a relatively larger amount of material was eluted at greater volumes. There was only partial separation of the proteins on the column; the main peak contained β- and αs-caseins, their larger breakdown products and some para-κ-casein, and the third peak contained smaller breakdown products of the main casein fractions and para-κ-casein. Although the resolution was considerably inferior to that obtained by gel electrophoresis, the method has the advantage over this and other methods in that it gives a quantitative analysis of the sample based on molecular size. Thus, it is suggested that it may find use in analysing protein breakdown in cheese during ripening.

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Characterization of immunoreactive dynorphin in human phaeochromocytomas

Journal of Endocrinology ◽

10.1677/joe.0.1170123 ◽

1988 ◽

Vol 117 (1) ◽

pp. 123-132 ◽

Cited By ~ 8

Author(s):

T. A. Howlett ◽

G. M. Besser ◽

L. H. Rees

Keyword(s):

Acetic Acid ◽

Gel Filtration ◽

Molecular Size ◽

Major Peak ◽

Gel Filtration Chromatography ◽

Post Translational Modifications ◽

Minor Peak ◽

Wet Weight ◽

A Minor

ABSTRACT The prodynorphin-derived opioids, dynorphin (DYN) and α-neoendorphin (αNE) were studied in 24 human phaeochromocytomas and related tumours. Nineteen tumours, extracted in HCl (0·1 mol/l), contained concentrations of immunoreactive DYN (ir-DYN) ranging from < 0·5 to 794 pmol/g wet weight. None of the extracts in HCl contained ir-αNE (all < 2·4 pmol/g). Sephadex G-50 gel filtration chromatography of ir-DYN in HCl (0·1 mol/l) extracts of six tumours revealed three small peaks of ir-DYN of higher molecular size (approximately 12 000, 6000 and 3000 daltons), a minor peak of ir-DYN eluting just after DYN(1–17), and a broad major peak, consisting of at least three components, which was significantly retarded and eluted after the salt volume of the column. High-pressure liquid chromatography (HPLC) of these extracts revealed multiple peaks of ir-DYN, most of which did not coelute with any synthetic DYN peptides. On both gel filtration chromatography and HPLC, one of the minor peaks coeluted with DYN(1–32). None of the peaks of ir-DYN coeluted with DYN(1–17) which had been acetylated using acetic anhydride. Extracts of the same tumours in acetic acid (0·1 mol/l) yielded similar values for ir-DYN content, but parallelism in the assay was improved. Sephadex G-50 chromatography revealed a different pattern of ir-DYN with a major peak coeluting with DYN(1–17) and, in two tumours, a minor peak coeluting with DYN(1–8). Studies with HPLC revealed, however, that substantial degradation of synthetic DYN occurred during extraction in acetic acid (0·1 mol/l) in spite of the precautions taken. Phaeochromocytomas frequently contain ir-DYN in concentrations which may approach that of the mammalian pituitary. These tumours did not, however, contain ir-αNE and, with the possible exception of a small amount of DYN(1–32), the ir-DYN present did not correspond with any known sequences. Thus, whilst prodynorphin is expressed in phaeochromocytomas, it does not seem to be processed to the usual end-products, and post-translational modifications therefore seem likely. Enzymatic degradation of DYN may occur during extraction in acetic acid (0·1 mol/l), and this medium should, therefore, be avoided in studies of such labile peptides. J. Endocr. (1988) 117, 123–132

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Characterization of Native Human Thrombopoietin in the Blood of Normal Individuals and of Patients with Haematologic Disorders

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614624 ◽

1999 ◽

Vol 82 (07) ◽

pp. 24-29 ◽

Cited By ~ 8

Author(s):

Atsushi Matsumoto ◽

Tomoyuki Tahara ◽

Haruhiko Morita ◽

Kensuke Usuki ◽

Hideya Ohashi ◽

...

Keyword(s):

Size Distribution ◽

Gel Filtration ◽

Molecular Size ◽

Immunoblot Analysis ◽

Biologically Active ◽

Amino Acid Residues ◽

Normal Individuals ◽

Normal Human ◽

A Minor ◽

Two Peaks

SummaryThrombopoietin (TPO) isolated from thrombocytopenic plasma of various animal species has previously been shown to comprise only truncated forms of the molecule, presumably generated by proteolysis. Native TPO has now been partially purified from normal human plasma by immunoaffinity chromatography and was confirmed to be biologically active. Gel filtration in the presence of SDS revealed that TPO eluted in two peaks: a major peak corresponding to the elution position of fully glycosylated recombinant human TPO (rhTPO) consisting of 332 amino acid residues, and a minor peak corresponding to a smaller molecular size. Immunoblot analysis also revealed that most plasma-derived TPO migrated at the same position as fully glycosylated rhTPO, corresponding to a molecular size of ~80 to 100 kDa. Furthermore, the size distribution of circulating TPO in patients with various haematologic disorders did not differ markedly from that of plasma-derived TPO from healthy individuals. These results indicate that the truncation of circulating TPO is not related to disease pa-thophysiology, and that the predominant form of TPO in blood is a biologically active ~80- to 100-kDa species. The size distribution of TPO extracted from normal platelets was similar to that of TPO in plasma; the proportion of truncated TPO was decreased by prior incubation of platelets with hirudin, indicating that the endogenous truncated TPO, at least in platelet extract, was generated by thrombin-mediated cleavage.

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Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

Biochemical Journal ◽

10.1042/bj2820711 ◽

1992 ◽

Vol 282 (3) ◽

pp. 711-714 ◽

Cited By ~ 49

Author(s):

E Blée ◽

F Schuber

Keyword(s):

Glycine Max ◽

Gel Filtration ◽

Soluble Form ◽

Soluble Enzyme ◽

Purification And Characterization ◽

Epoxide Hydrolases ◽

Major Activity ◽

Apparent Homogeneity ◽

A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

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Ecological Effects of Perorally Administered Pivmecillinam on the Normal Vaginal Microflora

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.170-175.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 170-175 ◽

Cited By ~ 8

Author(s):

Åsa Sullivan ◽

Aino Fianu-Jonasson ◽

Britt-Marie Landgren ◽

Carl Erik Nord

Keyword(s):

Escherichia Coli ◽

Candida Albicans ◽

Antimicrobial Agents ◽

Ecological Impact ◽

Menstrual Cycles ◽

The Third ◽

Vaginal Microflora ◽

Normal Microflora ◽

Facultative Anaerobic ◽

A Minor

ABSTRACT The knowledge of the effects of antimicrobial agents on the normal vaginal microflora is limited. The objective of the present study was to study the ecological impact of pivmecillinam on the normal vaginal microflora. In 20 healthy women, the estimated day of ovulation was determined during three subsequent menstrual cycles. Microbiological and clinical examinations were performed on the estimated day of ovulation and on day 3 in all cycles and also on day 7 after ovulation in cycles 1 and 2. Anaerobic and facultative anaerobic gram-positive rods, mainly species of lactobacilli and actinomycetes, dominated the microflora. One woman was colonized on the third day of administration with a resistant Escherichia coli strain, and Candida albicans was detected in one woman on days 3 and 7 in cycle 2. No other major changes in the normal microflora occurred during the study. Administration of pivmecillinam had a minor ecological impact on the normal vaginal microflora.

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Effect of extractant and molecular size on the optical and chemical properties of soil humic acids

Soil Research ◽

10.1071/sr9690229 ◽

1969 ◽

Vol 7 (3) ◽

pp. 229 ◽

Cited By ~ 50

Author(s):

JHA Butler ◽

JN Ladd

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Humic Acids ◽

Gel Filtration ◽

Chemical Properties ◽

Molecular Size ◽

Carboxyl Content ◽

Peptide Bonds ◽

Molecular Weights ◽

Amino Acid Contents

Humic acids extracted from soil with sodium pyrophosphate have greater proportions of lower molecular weight material, less acid-hydrolysable amino acid nitrogen contents, but greater carboxyl contents and extinction values (260 and 450 nm) than humic acids extracted subsequently from the same sample with alkali. Humic acids extracted with alkali from fresh soil samples have intermediate values. Extinction values at 260 nm are directly correlated with carboxyl contents for a given soil. Different crop histories have no significant effect on the measured properties of the extracted humic acids. An alkali-extracted humic acid has been fractionated by gel filtration into seven fractions of different nominal molecular weight ranges. As the molecular weights of the fractions increase, both aliphatic C-H (based on infrared absorption at 2900 cm-1) and acid-hydrolysable amino acid contents increase, whereas extinction values at 260 nm and carboxyl contents decrease. The infrared spectra of the high molecular weight fractions have peaks at 1650 and 1510 cm-1 which correlate with acid-hydrolysable amino acid contents and which correspond to amide I and II bands of peptide bonds. Alkaline hydrolysis to split peptide bonds eliminates both these peaks. The spectra also have peaks at 1720 and 1210 cm-1 which correlate with the carboxyl content.

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Rat liver alcohol dehydrogenase. Purification and properties

Biochemical Journal ◽

10.1042/bj1251039 ◽

1971 ◽

Vol 125 (4) ◽

pp. 1039-1047 ◽

Cited By ~ 69

Author(s):

M J Arslanian ◽

E Pascoe ◽

J G Reinhold

Keyword(s):

Molecular Weight ◽

Alcohol Dehydrogenase ◽

Rat Liver ◽

Gel Filtration ◽

Rabbit Antiserum ◽

Minor Component ◽

Disc Electrophoresis ◽

Supernatant Fraction ◽

Liver Alcohol Dehydrogenase ◽

A Minor

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.

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Age-dependent changes in volume and haemoglobin content of erythrocytes in the carp (Cyprinus carpio L.)

Journal of Experimental Biology ◽

10.1242/jeb.141.1.133 ◽

1989 ◽

Vol 141 (1) ◽

pp. 133-149 ◽

Cited By ~ 2

Author(s):

W. Speckner ◽

J. F. Schindler ◽

C. Albers

Keyword(s):

Gel Filtration ◽

Linear Increase ◽

Human Erythrocytes ◽

Main Peak ◽

Red Cell ◽

Human Haemoglobin ◽

Cell Fractions ◽

Haemoglobin Content

Carp erythrocytes were fractionated by angle-head centrifugation which yielded fractions with a linear increase in density. Haematological examinations revealed that the heavier red blood cells of carp had greater volumes (MCV), more haemoglobin (MCH) and higher haemoglobin concentrations (MCHC) than light ones. The same experiments with human red cell fractions yielded a decrease in MCV, constant MCH and an increase in MCHC. Haemoglobin content in individual erythrocytes was also determined by scanning stage absorbance cytophotometry to establish the frequency distribution of the cellular haemoglobin contents. In carp, the distribution was symmetrical with the means increasing with density. No such change with cell density was found in human erythrocytes. Both carp and human erythrocytes incorporated [2-14C]glycine in vitro. After gel filtration, radioactivity was detected in carp, but not in human, haemoglobin fractions. 14C was found in all three haemoglobin fractions, obtained by isoelectric focusing, and was present in the haem and in the globin. [2-14C]glycine-labelled erythrocytes were reinjected into chronically cannulated carp and followed in vivo for several months. With time, the main peak of scintillation counts shifted from red cell fractions of low to high density. This is considered as evidence that density and age of red cells in carp are positively correlated and that erythrocytes can synthesize haemoglobin while circulating in the peripheral blood.

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The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

Biochemical Journal ◽

10.1042/bj1300211 ◽

1972 ◽

Vol 130 (1) ◽

pp. 211-219 ◽

Cited By ~ 14

Author(s):

Colin H. Self ◽

P. David J. Weitzman

Keyword(s):

Molecular Weight ◽

Ph Dependence ◽

Gel Filtration ◽

Deae Cellulose ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Molecular Size ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Low Concentrations ◽

Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

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THE EFFECT OF LONG-TERM CORTICOTROPHIN TREATMENT ON THE CORTICOSTEROID-BINDING CAPACITY OF TRANSCORTIN

Journal of Endocrinology ◽

10.1677/joe.0.0390565 ◽

1967 ◽

Vol 39 (4) ◽

pp. 565-569 ◽

Cited By ~ 7

Author(s):

ZSUZSANNA ÁCS ◽

E. STARK ◽

L. CSÁKI

Keyword(s):

Binding Capacity ◽

Gel Filtration ◽

Filtration Method

SUMMARY In rats consecutive daily doses of corticotrophin significantly reduced the corticosterone-binding capacity of transcortin as measured by the gel filtration method, even after previous removal of the adrenals or ovaries. Treatment with formalin for 14 days produced similar, though slightly lesser, changes.

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