Quantitative studies of rapid polymerization equilibrium by gel filtration. The z-average elution volume of a reaction boundary

1967 ◽  
Vol 71 (13) ◽  
pp. 4492-4498 ◽  
Author(s):  
D. J. Winzor ◽  
Joan P. Loke ◽  
Lawrence W. Nichol
Keyword(s):  
Gel Filtration ◽  
Elution Volume ◽  
Quantitative Studies ◽  
Polymerization Equilibrium

A New Case Of Dysfibrinogenemia : Isolation Of The Abnormal, Unclottable Fibrinogen Population

1981 ◽  
Author(s):  
M Jandrot-Perrus ◽  
M H Aurousseau ◽  
F Josso
Keyword(s):  
Factor Xiii ◽  
Gel Filtration ◽  
Healthy Woman ◽  
Fibrin Clot ◽  
Elution Volume ◽  
Functional Studies ◽  
Sds Page ◽  
Normal Functional ◽  
A Chain ◽  
And Control

In a 81-year-old healthy woman, gross abnormalities of fibrin formation in routine tests led to the discovery of a dysfibrinogenemia.Abnormal and control fibrinogens were purified in parallel using precipitation by glycine (Kazal) ; final clottability was 95-98 % for the control and 50 % for the patient’s fibrinogen. Electrophoretic behaviour of the fibrinogen momecule, the three chains and the products of fibrin cross-linking by factor XIII a was normal. Functional studies gave the following results : (i) delayed coagulation by thrombin, Reptilase and Venacil with gross abnormalities of the clot; (ii) inhibition of coagulation of normal fibrinogen ; (iii) poor fibrin monomer aggregation (opacimetry) ; (iv) delayed fibrinogen proteolysis by plasmin (SDS-PAGE) . Release of fibrinopeptide A by thrombin was incomplete (RIA).Fibrinogen NH2-terminal residues were found normal, but the presence of ALA-residue in fibrin clot and in the supernatant showed that part of fibrinogen was not clotted, either copolymerized with fibrin or remaining in solution. Gel filtration of the supernatant showed the presence of both soluble complexes and fibrinogen characterized by the elution volume of the peak and NH2-terminal analysis. This fibrinogen population was unclottable by thrombin and inhibited clotting of normal fibrinogen.These preliminary results suggest the existence of a defect on the A-a chain of this abnormal fibrinogen which was called fibrinogen Bondy.

Isolation of the γc-globulin of bovine cerebrospinal fluid

1968 ◽  
Vol 46 (3) ◽  
pp. 273-276
Author(s):  
Catherine F. C. MacPherson ◽  
François Feldmuller
Keyword(s):  
Molecular Weight ◽  
Cerebrospinal Fluid ◽  
Gel Filtration ◽  
Globulin Fraction ◽  
Elution Volume ◽  
Immunological Activity ◽  
Immunochemical Method ◽  
Milk Whey ◽  
Preliminary Filtration ◽  
Gel Diffusion

The γc-globulin of bovine cerebrospinal fluid (CSF) was isolated in immunologically pure form from the globulin fraction of CSF or milk whey by gel filtration on Sephadex G-75. The globulin fraction was obtained by precipitation with sodium or ammonium sulfate rather than by DEAE chromatography because salt precipitation resulted in greater yields of the γc-globulin. When the protein was isolated from bovine colostral whey, about 90% of the immunoglobulin G (IgG) globulin was removed by a preliminary filtration of the whey on Sephadex G-75. The fraction containing the γc-globulin admixed with IgG globulin was then re-chromatographed on Sephadex G-75 to remove the IgG globulin. On gel filtration through a column of Sephadex G-75 that had been calibrated with proteins of known molecular weight, the elution volume of the γc-globulin corresponded to a molecular weight of 30 000. Different lots of γc-globulin that contained no detectable impurities by gel-diffusion tests were compared on a weight basis by a quantitative immunochemical method, and were found to have equal immunological activity.

Studies on the Dissociation of Porcine Haptoglobin

1972 ◽  
Vol 50 (7) ◽  
pp. 775-781 ◽  
Author(s):  
W. L. Lockhart ◽  
W. P. Chung ◽  
David B. Smith
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Light Chains ◽  
Dilute Solution ◽  
Disulfide Bridges ◽  
Elution Volume ◽  
Reversible Dissociation ◽  
Electrophoretic Mobilities ◽  
Chain Composition

Porcine haptoglobin was tested for reversible dissociation in dilute solution by gel filtration. Elution volume in Bio-Gel P-150 was independent of concentration and shapes of elution profiles failed to show dissociation. Molecular weight in dilute solution was estimated as 96 500. Interchain disulfide bridges were assayed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Partially reduced samples showed a series of intermediates but fully reduced samples showed only heavy and light chains. Intermediates were tentatively identified as to chain composition from their electrophoretic mobilities.

Molecular Size Distribution of Fibrinogen Derivatives Formed in Vitro and in Vivo: a Chromatographic Study

1976 ◽  
Vol 36 (01) ◽  
pp. 014-026 ◽  
Author(s):  
M. B Donati ◽  
R Verhaeghe ◽  
D. E Culasso ◽  
J Vermylen
Keyword(s):  
Biological Fluids ◽  
Gel Filtration ◽  
Molecular Size ◽  
Gel Chromatography ◽  
Chromatographic Study ◽  
Elution Volume ◽  
Human Fibrinogen ◽  
Elution Pattern

SummaryUsing gel chromatography, fibrinogen derivatives present in purified systems or in biological fluids were separated and partially characterized. Eight groups of fibrinogen derivatives could be separated by gel filtration through 6% agarose in large columns, four with an elution volume smaller and four groups with an elution volume larger than that of fibrinogen. Careful calibration of the column allowed estimation of the diffusion coefficients of some of the derivatives and, thus, comparison with derivatives previously identified. Three, rather than two, groups of intermediate derivatives were observed during the degradation of human fibrinogen by plasmin in vitro or in vivo. One of these had a marked tendency to polymerize.A rather distinct difference in elution pattern was found between plasma obtained during streptokinase administration and from patients with intravascular coagulation.

Comparison Of Naturally Occurring And CaCl2-Separated Canine Factor VIII-Coagulants Free Of FACTOR VIII-Related Antigen

1981 ◽  
Author(s):  
R E Benson ◽  
W J Dodds
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Room Temperature ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Lower Molecular Weight ◽  
Elution Volume ◽  
Stable Factor ◽  
Naturally Occurring ◽  
Similar Combination

Previous studies demonstrated that plasma from dogs homozygous for von Willebrand’s disease (VWD) without detectable factor VUI-related antigen (VIII:RAg) contained moderate levels of a stable factor VIH-coagulant (VIII:C), which had lower apparent molecular weight and combined with the VIII:RAg of canine hemophilic plasma. We have now compared this VWD-VIII:C to the CaCl2-separated form of VIII:C prepared from normal canine factor VIII. The VWD plasma was fractionated at room temperature by 6% agarose gel filtration in 0.15M NaCl and 0.24M CaCl2 buffers, pH 7.25; the VIII:C eluted in each buffer with a relative elution volume (Ve/Vo) of 1.4. Isolated lower molecular weight VIII:C stabilized with albumin (5 mg/ml) and dialysed free of Ca++ was prepared from normal purified canine factor VIII by elution in 0.24M CaCl2 buffer. This CaCl2~separated VIII:C was rechromatographed in both buffers as above and had a Ve/Vo of 1.9. When the VWD and CaCl2 forms of VIII:C were each combined with canine hemophilic plasma and gel filtered in 0.15M NaCl buffer, the VIII:C’s now appeared at the void volume. These mixtures with 1/10 volume 2.4M CaCl2 added before or after combination were also analyzed in 0.24M CaCl2 buffer. The CaCl2-VIII:C eluted with a Ve/Vo of 1.9 regardless of the order of 2.4M CaCl2 addition, but the VWD-VIII:C eluted with Ve/Vo’s of 1.4 when CaCl2 was added before mixing and 1.9 when added afterwards.Thus, when mixed with hemophilic plasmas both the CaCl2 and VWD forms of VIII:C exhibited similar combination and separation characteristics. However, the finding that the CaCl2-VIII:C is of apparent lower molecular weight than the VWD form suggests that the CaCl2-separated VIII:C is different from the naturally occurring form of VWD (VIII: RAg-free) VIII:C.

scholarly journals Purification and properties of bovine pituitary follitropin

1976 ◽  
Vol 159 (3) ◽  
pp. 651-659 ◽  
Author(s):  
K W Cheng
Keyword(s):  
Amino Acid ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Elution Volume ◽  
Purification And Properties ◽  
Receptor Assay ◽  
Ion Exchange Column ◽  
Pituitary Glands ◽  
Radioligand Receptor Assay

A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine.

Methods for studying the binding of aluminum by serum protein.

1985 ◽  
Vol 31 (12) ◽  
pp. 1969-1973 ◽  
Author(s):  
H Rahman ◽  
A W Skillen ◽  
S M Channon ◽  
M K Ward ◽  
D N Kerr
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Affinity Chromatography ◽  
Serum Protein ◽  
Reliable Method ◽  
Binding Protein ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Quantitative Studies

Abstract We describe methods for studying the binding of Al by protein in serum: ultrafiltration, gel filtration, and immuno-affinity chromatography. For ultrafiltration we used an Amicon YM10 cellophane membrane with a nominal cutoff of 10 000 Da to separate ultrafiltrable and non-ultrafiltrable Al. For gel filtration we used Sephacryl S-300, and for immuno-affinity chromatography we used anti-transferrin coupled to CNBr-activated Sepharose to identify the Al-binding protein. For 30 normal subjects 54% of the total Al in serum was non-ultrafiltrable; for 30 patients with chronic renal failure being treated by hemodialysis 67% was non-ultrafiltrable. In both groups transferrin was identified as the major Al-binding protein in the serum. Results of gel-filtration studies should be interpreted with caution: some gel media adsorb "free" Al, which can be subsequently taken up by transferrin or desferrioxamine passing through the column. We find affinity chromatography to be a specific and reliable method, suitable for use in quantitative studies.

DISCREPANCIES IN PLASMA LH ACTIVITIES AS MEASURED BY RADIOIMMUNOASSAY AND AN IN VITRO BIOASSAY

1974 ◽  
Vol 77 (4) ◽  
pp. 672-685 ◽  
Author(s):  
M. H. Qazi ◽  
P. Romaní ◽  
E. Diczfalusy
Keyword(s):  
Molecular Weight ◽  
Biological Activity ◽  
Gel Filtration ◽  
Biologically Active ◽  
Plasma Samples ◽  
Elution Volume ◽  
Immunological Activity ◽  
Molecular Weight Range ◽  
Weight Range

ABSTRACT Using a highly sensitive in vitro bioassay system, luteinizing hormone activity has been measured in parallel with radioimmunoassays in postmenopausal plasma and plasma obtained from women in various phases of the menstrual cycle (follicular, mid-cycle, luteal). Biological and immunological activities were measured directly in plasma samples without any chemical manipulation. The biological activity (B) was always higher than the immunological activity (I); the B/I ratio varied from 2.1 to 14.0. Gel filtration of pooled plasma samples through Sephadex G-100 revealed major discrepancies in each physiological state when immunological and biological activities were measured in each fraction. The biological activity was eluted as a single peak behind the elution volume of bovine serum albumin, but in front of the elution volume of chymotrypsinogen. It was invariably preceded by a small hump. The immunological activity was spread all over the chromatogram. Areas of immunological activity without any biological activity were located on either side of the biologically active fractions, both in the high molecular weight range (including the void volume) and in the low molecular weight range. The biological LH activity recovered following fractionation on Sephadex G-100 was in close agreement with that loaded, whereas the immunological activity recovered following gel filtration exceeded the loaded activity by a factor of 6–8. In the various physiological states, 11 to 44 % of the total immunological activity recovered was not associated with any biological activity. Furthermore, there was a marked variation in the ratio of biological to immunological activities of those fractions which contained biological activity. It is suggested that the specificity of current RIA methods could be improved significantly by preparing antisera which react only with biologically active LH species.

The Effect of Guanidinium Chloride on the Self-Association of Bovine Liver Glutamate Dehydrogenase: A Gel Filtration Study

1987 ◽  
Vol 42 (3) ◽  
pp. 217-220
Author(s):  
Alberto Mazzini ◽  
Roberto Favilla
Keyword(s):  
Sodium Chloride ◽  
Glutamate Dehydrogenase ◽  
Gel Filtration ◽  
Bovine Liver ◽  
The Self ◽  
Gel Chromatography ◽  
Elution Volume ◽  
Guanidinium Chloride ◽  
Trimer Formation ◽  
Self Association

The associative behaviour of bovine liver glutamate dehydrogenase has been studied by gel chromatography at neutral pH in 1 ᴍ guanidinium chloride and 1 ᴍ sodium chloride. In guanidinium chloride both the elution volume and the elution profile of the enzyme are independ­ent of protein concentration, whereas in sodium chloride they are strongly dependent on it. In NaCl the enzyme behaves as expected according to the well established random association model, whereas in guanidinium chloride it appears to have completely lost the self-associative property. Furthermore, since the elution volume of the enzyme in guanidinium chloride corre­sponds to that of an hexamer, trimer formation reported to occur in these conditions is not confirmed by this technique.

Sign in / Sign up

Export Citation Format

Share Document