Molecular weight of erythropoietin from anemic sheep plasma and human urine

1968 ◽  
Vol 46 (8) ◽  
pp. 731-734 ◽  
Author(s):  
W. Lukowsky ◽  
R. H. Painter
Keyword(s):  
Molecular Weight ◽  
Human Urine ◽  
Good Yield ◽  
Gel Filtration ◽  
Single Component ◽  
Elution Volume

The properties of erythropoietin from sheep plasma and human urine were compared by gel filtration in columns of Sephadex G-150 calibrated with proteins of known molecular weight. Erythropoietin activity eluted as a single component in good yield at an elution volume consistent with a molecular weight of 60 000 in each case. No activity was detected in other fractions of the eluate, indicating the homogeneity of erythropoietin from both sources.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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.

IMMUNOREACTIVE GROWTH HORMONE IN HUMAN URINE

1972 ◽  
Vol 71 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Kristian F. Hanssen
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Linear Relationship ◽  
Human Urine ◽  
Gel Filtration ◽  
Human Growth ◽  
Normal Subjects ◽  
Normal Human ◽  
Radio Immunoassay

ABSTRACT By using a double antibody radio-immunoassay (pre-precipitation technique) for the determination of immunoreactive human growth hormone (IRHGH) in normal human urine concentrated by dialysis and lyophilization, a factor was revealed that displaces 125I-HGH from HGH antibodies. This displacement was neither due to salts nor to glucose; it is suggested that it is due to IRHGH in the urine. A linear relationship between dilution of urine and the measured IRHGH concentration was obtained. Recovery of exogenous HGH was between 70–105%. The recovery of IRHGH from different volumes of urine following dialysis and lyophilization was between 97–110%. Plasma IRHGH and urinary IRHGH was measured simultaneously after HGH injection in a normal subject. A correlation was shown between plasma IRHGH and urinary IRHGH. In 9 normal subjects, the urinary IRHGH ranged from 28–53 ng/24 h. The excretion of urinary IRHGH was increased in acromegaly and was diminished in some, but not in all patients with adult hypopituitarism. The urinary IRHGH was further studied by gel filtration. It was recovered in one peak corresponding to a molecular weight of approximately 20 000 – 30 000. However, in the present work it was not clarified whether the urinary IRHGH represents pituitary HGH excreted in the urine or a metabolite of high molecular weight with retained immunological properties.

scholarly journals Plasmin Inhibitors in Human Urine

1977 ◽  
Author(s):  
H. Sumi ◽  
Y. Takada ◽  
A. Takada
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Molecular Form ◽  
Membrane Filter ◽  
Gel Filtration ◽  
Total Activity ◽  
Native Form ◽  
Molecular Weights ◽  
Two Peaks

By the gel filtration of urinary protein on Sephadex G-200, two peaks of activity to hydrolyze Nα-acetylglycyl-L-lysine methyl ester (AGLMe) were detected. One was the native form of urokinase, and the molecular weight was about 54,000. The other was of high molecular weight, and eluted in void fraction. The high molecular form was thought to be a complex of urokinase and urinary plasmin inhibitor (UPI). By using Arg-Sepharose, membrane filter (M.W. 10,000), and pevikon block electrophoresis, we could isolate four types of UPI from normal human urine. One UPI was positively charged at pH 8.6, and of high molecular weight. Other types were negatively charged, and the molecular weights by gel filtration on Sephadex G-200 were about 67,000 (UPI6.7), 45,000 (UPI4.5), and 22, 000 (UPI2.2), respectively. In acrylamide disc gel electrophoresis, UPI57 migrated to serum prealbumin fraction, and UPI45 and UPI? 2 were less anionic. Negative-charged UPIs could be adsorbed on trypsin-Sepharose, and were thought to be identical to urinary trypsin inhibitors. Purified UPIs showed strong inhibition on caseinolytic- and esterolytic-activities of plasmin, and the total activity was about 16 UPIU(inhibited 16 casein U of plasmin)/liter of urine.

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.

Purification of High Molecular Weight Urokinase from Human Urine and Comparative Study of Two Active Forms of Urokinase

1983 ◽  
Vol 49 (02) ◽  
pp. 091-095 ◽  
Author(s):  
Takeji Shibatani ◽  
Toshio Kakimoto ◽  
Ichiro Chibata
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Improved Method ◽  
Assay Methods ◽  
Hydroxyl Apatite ◽  
Cellulose Column ◽  
Molecular Weight Form

SummaryAn improved method for the purification of high molecular weight urokinase to homogeneity from human urine was established. A yield of 32% with a 3,100-fold purification was obtained by Hyflo Super-Cel treatment, heat treatment at 60° C for 10 hr, serial column chromatography on DEAE-Sepharose CL-6B and 0-[3-(p-sulfophenylamino)-2-hydroxypropyl]-cellulose (SFOP-cellulose), and gel filtration on Ultrogel AcA 54. The low molecular weight form of urokinase was also purified to homogeneity by chromatography on hydroxyl apatite and gel filtration on Sephadex G-75 after the SFOP-cellulose column step. The high molecular weight urokinase had only one isoelectric form with a pi of 9.7, whereas the low molecular weight form had six isoelectric subforms with pi values between 9.4 and 6.4. The absorption coefficients at 280 nm of both urokinase forms were 13.61 and 13.50, respectively. Fibrinolytic and esterolytic activities of the two urokinase forms were compared in various assay methods.

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.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Separation of Plasminogen Activators from Human Uterine Tissue and a Comparison with Activators from Human Urine and Porcine Tissue

1979 ◽  
Vol 41 (04) ◽  
pp. 718-733 ◽  
Author(s):  
Preben Kok
Keyword(s):  
Plasminogen Activator ◽  
Human Urine ◽  
Gel Filtration ◽  
Ammonium Thiocyanate ◽  
Plasminogen Activators ◽  
Particle Sizes ◽  
Uterine Tissue ◽  
Porcine Tissue ◽  
Major Activity ◽  
Proliferative Phase

SummaryThree types of plasminogen activator could be distinguished in extracts from human uterine tissue. The activators differed in thermostability or in mode of inhibition by EACA.All the extracts contained stable as well as labile activators. The saline extracts were uniformly inhibited by increasing concentrations of EACA. Extracts made with 2 M ammonium thiocyanate were either uniformly inhibited by EACA or showed deflections indicating contamination with an activator, which was inhibited in a biphasic manner. It was possible to distinguish between: (1) An activator, abundantly present in the tissue, which was uniformly inhibited and stable. (2) Another uniformly inhibited activator, which was labile. (3) An activator, inhibited in a biphasic manner, similar to urokinase, which was present in varying amounts in uteri with the endometrium in the proliferative phase.Gel filtration of the uterine extracts showed two major activity peaks corresponding to particle sizes of 60,000 dalton and about 10,000 dalton.Antiserum to purified plasminogen activator, prepared from porcine ovaries, inhibited the activity of the human uterine extracts, but not the activities of human urokinase or urine. Urokinase antiserum in a concentration completely inhibiting human urine or urokinase, inhibited only 10% or less of the activities of human uterine extracts.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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