Cohn Fraction IV-1 Research Articles

Purification of a basic somatomedin, from human plasma Cohn fraction IV-1, with physicochemical and radioimmunoassay similarity to somatomedin-C and insulin-like growth factor.

A basic somatomedin (SM) was purified from human plasma Cohn fraction IV-1 using an initial acid--ethanol--acetone extraction procedure followed by alternating molecular size or charge protein separation techniques. The final recovery of SM bioactivity was approximately 2% of that present in the starting Cohn fraction. The purified SM has an approximate molecular weight of 7500, pI 8.6, 4000 SM bioactivity units per milligram (as measured by a hypophysectomized rat bioassay) and a parallel approximately equipotent radioimmunoassay dose--response curve to SM-C and insulin-like growth factor-1 (IGF-I). Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of this purified SM revealed a single protein band. The preliminary determination of the amino acid sequence of the N terminus suggested that this SM preparation was over 75% pure and the first five N-terminal amino acids were identical with those of IGF-I.

Urinary and plasma testosterone glucosiduronate measurement by a simple ria method

A simple, specific and rapid radioimmunoassay (RIA) for the determination of testosterone-17β-glucosi-duronate (TG) in urine and plasma is described. Antibodies against TG were raised in rabbits after injection of an immunogenic complex obtained by condensation of Cohn's fraction IV-4 (CF) and TG. This complex revealed higher titer developing than those obtained by simultaneous immunization with another TG complex using bovine serum albumin as carrier protein. To separate free and bound fractions, three methods were compared: Charcoal adsorption, Polyethyleneglycol precipitation and Hydroxyapatite separation. Hydroxyapatite showed a good coefficient of variation (intra-assay 6%, and inter-assay 11%) together with the possibility of working at room temperature and reducing counting cost. TG was estimated in small aliquots of male and female urine after evaporating overnight at 50°C in order to eliminate interfering material. Plasma samples were pre-extracted with diethylether to remove non-conjugated steroids and extracted again with ethanol. The mean excretion of TG in the urine of 90 healthy men amounted to 178 ± 46 μg/24 h and of 50 women to 31 ± 11 μg/24 h. In 56 women suffering from idiopathic hirsutism a mean TG of 68 ± 27 μg/24 h and in 40 men with hypogonadism 58 ± 27 μg/24 h were found. In order to avoid 24 h urine collections, a TG/creatinine index in early morning urine was tested. This showed good correlation ( r = 0.8) in both sexes with the correspondent daily excretion. Plasma TG concentrations were found to be 2.1 ± 0.45 ng/ml in 20 normal males, 0.47 ± 0.18 ng/ml in 20 normal females, 1.08 ± 0.3 in 9 hypodogonadal males and 1.36 ± 0.68 in 28 hirsute females. TG concentrations in testicular vein was 4.02 ± 1.4 ng/ml in 3 normal males (basal) and 5 ± 2.7 ng/ml 30 minutes after 500 IU HCG + 66 IU FSH, 65 IU LH (HMG) infusion. The simultaneous cubital vein values were 3 ± 1.3 ng/ml and 3.6 ± 2.3 ng/ml respectively. The method makes possible the determination of 300 urine samples or 120 plasma samples per week by a well trained technician.

Development of large scale fractionation methods. VII. Preparation of antithrombin III concentrate.

A large scale method for preparation of antithrombin III (AT III) concentrate from plasma or from Cohn fraction IV-1 (Fr. IV-1) has been described. It consists of the following steps: (a) partial purification by precipitation of impurities with 20% polyethylene glycol (PEG) 4000; (b) isolation of AT III from the PEG supernatant by batch adsorption and elution on heparin-Sepharose at a ratio corresponding to 45 vol of plasma or 80 vol of 10% Fr. IV-1 solution to 1 vol of gel; (c) concentration and desalting of the eluted AT III on a Pellicon ultrafiltration system; (d) pasteurization of AT III concentrate by heating for 10 h at 60 degrees C in the presence of 0.5 M sodium citrate at pH 7.5; (e) removal of excess citrate by gel filtration on Sephadex G-50; and (f) sterile filtration, filling and lyophilization. The recovery by activity was 32% from a 113-liter plasma batch and 16% from a 42-kg Fr. IV-1 batch. Both AT III concentrates, derived either from plasma or from Fr. IV-1, had similar specific activity and electrophoretic purity, were nonpyrogenic and met all other FDA requirements for biologic products. Pasteurization induced changes in disc gel and isotachophoretic patterns of AT III preparations.

Development of Large Scale Fractionation Methods

A large scale method for preparation of antithrombin III (AT III) concentrate from plasma or from Cohn fraction IV-1 (Fr. IV-1) has been described. It consists of the following steps: (a) partial purification by precipitation of impurities with 20% polyethylene glycol (PEG) 4000; (b) isolation of AT III from the PEG supernatant by batch adsorption and elution on heparin-Sepharose at a ratio corresponding to 45 voi of plasma or 80 voi of 10% Fr. IV-1 solution to 1 voi of gel; (c) concentration and desalting of the eluted AT III on a Pellicon ultrafiltration system; (d) pasteurization of AT III concentrate by heating for 10 h at 60 °C in the presence of 0.5 M sodium citrate at pH 7.5; (e) removal of excess citrate by gel filtration on Sephadex G-50; and (f) sterile filtration, filling and lyophilization. The recovery by activity was 32% from a 113-liter plasma batch and 16% from a 42-kg Fr. IV-1 batch. Both AT III concentrates, derived either from plasma or from Fr. IV-1, had similar specific activity and electrophoretic purity, were nonpyrogenic and met all other FDA requirements for biologic products. Pasteurization induced changes in disc gel and isotachophoretic patterns of AT III preparations.

Partial Purification, Characterization, and Assay of a Slightly Acidic Insulin-Like Peptide (ILAs) from Human Plasma*

An insulin radioreceptor assay (RRA) using human placental microsomal membranes was used to measure insulin-like activity (ILA) extracted from human plasma concentrates (Cohn fraction IV-4) by acid ethanol. The soluble activity (ILAs), chromatographed on Sephadex G-75 in 1 M acetic acid, migrated as a small molecule (fractional elution volume, 0.56) ahead of insulin (fractional elution volume, 0.70), whereas at neutral pH, ILAs migrated as a large molecular weight species. The ILAs peak from acid gel filtration on Sephadex was further purified by chromatography on carboxymethyl cellulose (CMC). The ILAs peak from both Sephadex and CMC diluted parallel to the porcine insulin standard in the insulin RRA and was totally unreactive in an insulin RIA. The CMC-purified material was iodinated and purified by binding to and elution from human placental membranes. The binding of [125I]ILAs to human placental membranes was inhibited only minimally by insulin and proinsulin and not at all by epidermal growth factor, nerve growth factor, glucagon, or lactogenic hormones, including human growth hormone. Multiplication-stimulating activity (MSA) inhibited in a manner parallel to ILAs. A Scatchard plot of the binding data was nonlinear. Sephadex ILAs was subjected to isoelectric focusing. The fractions assayed in both insulin and ILAs RRAs yielded comparable results. Peaks of ILA were observed at pHs 5.3, 6.6, and 8.4. When CMC-ILA was subjected to isoelectric focusing in polyacrylamide, a single peak of activity migrating between pH 6.2-6.8 was seen. [125I]ILAs focused at exactly the same pH. Electrophoresis of CMC-ILAs in acid-urea revealed a sharp peak of activity migrating with one of the five protein bands seen after staining. Again, [125I]ILAs comigrated with unlabeled ILAs. The molecular weight of ILAs, as determined on a calibrated Sephadex G-150 column at neutral pH, was 9,000-10,000 daltons. CMC-ILAs stimulated [14C]glucose incorporation into triglycerides of rat adipose tissue and augmented [3H]thymidine incorporation into human fibroblasts, chicken embryo fibroblasts, and BALB 3T3 cells as well as [35S]sulfate incorporation into macromolecules of rabbit chondrocyte culture medium. In summary, ILAs isolated on the basis of a RRA for insulin is a slightly acidic peptide with some of the biological activities expected of a somatomedin.

THE DETERMINATION OF 5α-ANDROSTANE-3α,17β-DIOL IN HUMAN PLASMA BY RADIOIMMUNOASSAY

Antibodies have been raised in rabbits against 3alpha, 17beta-dihydroxy-5alpha-androstane-6-0-carboxymethyloxime coupled with Cohn's fraction IV-4. The antiserum exhibited significant cross reactions with 5beta-androstane-3alph1, 17beta-diol, 5alpha-dihydrotestosterone, and testosterone. No cross reactions were observed with 5alpha-androstane-3beta,17beta-diol and 5-androstene-3beta,17beta-diol. The methodological criteria for the measurement of 5alpha-androstane-3alpha, 17beta-diol in human plasma were as follows: The specificity was ensured by separating the cross reacting steroids by thin layer chromatography. The intraassay and interassay coefficients of variation were found to be 6.2 and 10.2%, respectively. The sensitivity was 30 pg. The recovery of different amounts of 5alpha-androstane-3alpha,17beta-diol added to human plasma (80, 120, and 200 pg) yielded 91.3, 92.5, and 93.5%, respectively. The following concentrations of 5alpha-androstane-3alpha,17beta-diol have been determined in human plasma (mean +/- SD, ng/dl): Normal males: 18.98 +/- 5.9; normal females: 2.65 +/- 0.27; females with idiopathic hirsutism: 11.9 +/- 6.4; prepubertal children: not detectable.

Nelson-Town anti virus theory

For any virus that causes a biological change in the body, there is a substance that will prevent this virus from causing this biological change. We will call this substance an anti virus substance. Reasons for proposing this theory along with reasons for the prediction of 3 properties of an anti virus will be presented. Also to be presented is an anti virus circular diagram, a mechanism where when vitamin C is present the body can more easily manufacture the anti virus, other mechanisms, and the comparison of properties of humoral antibodies and interferon with suspected properties of anti viruses. 1. Town Fraction 2. Rheumatoid Arthritis 3. Anti Virus 4. Cohn Fraction IV-1 5. Leucocyte Lysosome Stabilization 6. Copper II 7. PPM Mechanism 8. Folic Acid Mechanism 9. Aspirin Mechanism 10. Humoral Antibodies 11. Interferon 12. Anti Virus Circular Diagram 1. Arthritis, Rheumatoid 2. Alpha Globulins 3. Copper 4. Lysosomes 5. Ceruloplasmin 6. Viral Interference 7. Ascorbic Acid

Pilot scale purification of α-galactosidase A from Cohn Fraction IV-1 of human plasma

Human plasma α-galactosidase A (α-D-galactoside galactohydrolase, EC 3.2.1.22) was purified 7000-fold over plasma levels from Cohn Fraction IV-1. The yield per kg starting material averaged 11 000 units (nmol galactose liberated per h) and the specifc activity was about 600 units per mg protein with 4-methylumbelliferyl-α-D-galactoside. The ratio of 4-methylumbelliferyl-α-galactosidase to ceramide trihexosidase activities was 6.2. Both activities were heat labile and exhibited the same relative mobilities on polyacrylamide gel electrophoresis. Enzymatic activity was stable for at least 4 months at 4 and −20°C. The endotoxin concentration of this preparation averaged 0.26 mg per mg protein.

Thymic hormone-like restoration by human prealbumin of azathioprine sensitivity of spleen cells from thymectomized mice.

Gel filtration of fresh human serum revealed that over 95% of the thymic-hormone-like activity was present in the fraction representing the total albumin and prealbumin. Further studies demonstrated that the activity resided in the prealbumin fraction; albumin was inactive. Prealbumin was isolated from Cohn fraction IV-1 of pooled human plasma by extraction with aqueous buffer, fractionation with (NH4)2SO4, gel filtration on Sephadex G-150, and preparative disc gel electrophoresis. The final product was homogeneous by analytical gel electrophoresis was 40,000 times more active than the starting material in an azathioprine-sensitive rosette assay. Physical and chemical characterization studies showed that the isolated product was identical to authentic human plasma prealbumin. Isolation of the prealbumin fraction from sera of adult thymectomized mice revealed that the rosette activity was substantially lower than that isolated from sera of normal mice, suggesting a thymic dependence of the prealbumin activity. In vitro and in vivo bioassays of the fraction obtained prior to the final step of the purification procedure support the conclusion that prealbumin exhibits thymic hormone-like activity.

Human plasma kallikrein: purification, enzyme characterization and quantitative determination in plasma.

Human plasma kallikrein was isolated from a plasma fraction related to Cohn fraction IV4 by affinity- and Sephadex G-150 chromotography yielding a material with 17.3 TAME-U/A280 unit. The preparation was characterized by immunological and enzymatic methods. Complex formation with alpha2-macroglobulin, C1-inactivator and aprotinin was demonstrated by immunoelectrophoresis. The bradykinin release from high-molecular weight kininogen and its inhibition by antibodies to kallikrein, AT III and AT III-heparin complex were measured using a biological test system (rat uterus). Time dependent inactivation of kallikrein by AT III, and AT III-heparin complex was shown by means of a synthetic kallikrein substrate: Bz-Pro-Phe-Arg-pNan. The same substrate was used to measure the activation of prekallikrein in plasma by kaolin and F XII a. Antibodies raised against kallikrein were shown to inhibit the reaction specifically. A quantitative determination of plasma prekallikrein by electroimmunodiffusion according to Laurell was developed: the plasma concentration in normal individuals was found to be 1.8 - 2.2 TAME-U/ml related to kallikrein activity; this corresponds approximately to 9 - 11 mg antigen/100 ml plasma.

Insulin, insulin-like activity and the human placenta

Specific insulin binding sites can be demonstrated in human placental membranes. Binding is readily reversible. Both the rates of binding and dissociation are temperature sensitive. Insulin analogues bind with affinities which parallel their biological activity. The affinity for insulin is of sufficient magnitude to bind physiologic levels of insulin. Insulin eluted from these binding sites is structurally intact. The binding sites are partially protein in composition and are found in the placental tissue of many species. The binding sites have the properties of biologically significant receptors in well-defined insulin target tissues. This suggests a direct role for insulin in regulating placental function. Two different soluble insulin degrading enzymes have been described in human placenta. In addition, a third membrane-associated degrading activity can be demonstrated. This enzyme was partially washed out of the membranes under circumstances which did not influence the insulin binding sites. This dissociation of degrading activity and binding suggests that they are not intimately related. An insulin radioreceptor assay (RRA) was used to follow the purification of an insulin-like peptide (ILAs, 7–12 × 10 3 daltons) from human Cohn fraction IV-4. ILAs had chemical and biological properties like those of somatomedin and NSILAs. It bound in a specific manner to a unique human placental receptor suggesting a role for it in placental function. Using this receptor and [ 125I]-ILAs, an ILAs RRA was developed and validated. The measurement of ILA in rat serum with the ILAs RRA revealed a large molecular weight crossreacting material whose level was greatly reduced by hypophysectomy. It has not been excluded that this material crossreacts in our assay through an ability to bind [ 125I]-ILAs. Further study must be aimed at characterizing the nature of insulin-like peptides in the circulation of animals and man.

Processes to increase the yields of normal serum albumin.

By heating in the presence of a stabilizer, albumin can be recovered from Cohn fraction IV-4 and also pyrogen-free from pyrogenic albumin.

Aphelenchoides rutgersi: Growth requirements for heme and purine nucleotides in crude chick embryo extract

The crude chick embryo extract (CEE) at 20% concentration (v/v) in the basal medium (M-10), enabled the growth, reproduction, and continuous culturing of Aphelenchoides rutgersi Hooper and Myers. No such growth promoting effect was found with chick embryo extract (Ultra filtrate), human gamma globulin and beta lipoprotein (Cohn fraction IV-4) when used either alone or in combination with hemin. The nematode growth factor(s) in the CEE were stable to trypsinization and temperature of 100 C for 5 min but not for 15 min. After acid treatment to pH 3.0 with sulfuric acid, the supernatant retained only partial activity. But, after treatment with perchloric acid to pH 3.0 or with activated charcoal, the CEE was completely ineffective as a growth supplement. The activity of charcoal-treated CEE has been completely recovered by addition of hemin (10 μg/ml M-10). Deletion of nucleic acid supplements from M-10 but not from the CEE did not affect the reproduction of the nematodes at all. However, the reproduction was significantly reduced when the nucleic acids were removed both from the M-10 and the CEE. Selective deletion of deoxyribonucleotides and purine-, and pyrimidinenucleotides of ribose series indicated only limited nutritional requirement for purine compounds.

A simple radioimmunoassay for the measurement of testosterone glucosiduronate in unextracted urine

A simple, reliable and rapid radioimmunoassay (RIA) for the determination of testosterone glucosiduronate (TG) in crude urine is described. Two protein-TG complexes were investigated in raising antibodies: a) Bovine serum albumin (BSA)-TG and b) human plasma Cohn's fraction IV-4 (CF)-TG. In rabbits, high liters of antibodies were obtained after the injection of CF-TG. The specificity of the antiserum was sufficiently high (cross reaction with free testosterone 27%, with 5α-dihydrotestosterone-glucosiduronate 20%). TG was estimated in small aliquots of male and female urine after evaporation overnight at 50° C in order to eliminate interfering material. The intraassay coefficient of varation (CV) was found to be 6% and the interassay CV 11%. TG has been determined in 40 samples of urine simultaneously by “direct” RIA and by a “classical” RIA following hydrolysis with β-glucuronidase. The coefficient of correlation was found to be 0.89. The mean excretion of TG in the urine of 26 healthy men amounted to 164 ± 51 μg/24 hours with a range from 97 to 546 μg/24 hours. In a group of 16 women a mean urinary excretion of TG of 24 ± 10 μg/24 hours was determined. The method allows a technician to assay 40 samples per day.

Characterization of renin-like activity in human plasma protein IV-4 fraction.

Cohn's fraction IV-4 of human plasma protein generates by itself a substance with slight pressor activity when incubted without any additives. This renin-like activity was readily inactivated by alkaline treatment. Furthermore, the generated pressor substance, when purified, was found very similar to [Aspl]-[Ile5]-angiotensin-I in the following characteristics; 1) heat-stability, 2) dializability, 3) inactivation by trypsin, 4) Rf value and 5) electrophoretic mobility at various pHs, 6) marked enhancement in pressor and oxytocic activities after incubation with normal human plasma or rabbit's lung extract, and 7) cross reactivity with [AspI]-[Ile5]-angiotensin-I in radioimmunoassay. It is concluded that renin-like activity in the preparation of fraction IV-4 of human plasma protein must be renin or an extremely similar enzyme.

Isolation and characterization of alpha-1-antitrypsin from the Cohn fraction IV-I of human plasma.

Abstract Cohn Fraction IV-I from pooled human plasma was used as a starting material in the large-scale purification of alpha-1-antitrypsin (alpha-1-AT). Following ion-exchange, blospecific affinity and gel exclusion chromatographic procedures, material of high biological activity was obtained in 307percnt; overall yield. Homogeneity was demonstrated by acrylamide gel electrophoresis, immunoelectrophoresis, ultracentrifugation, gel filtration and end-group determination. The present preparation should be applicable to large scale industrial processing of alpha-1-AT with the potential for use in protein replacement therapy.

Human plasma kallikrein: Purification and preliminary characterization

A method is described for the convenient purification of the protease plasma kallikrein from human Cohn fraction IV-1. The enzyme was produced by endogenous activation after acid treatment to remove an inhibitor and was concentrated by the successive use of affinity adsorbents prepared by the immobilization of soybean trypsin inhibitor and aminobenzamidine. The esterase- and kinin-producing activities were enriched about 1100-fold from fraction IV-1. Several properties of plasma kallikrein strengthen the impression that it is related to trypsin, namely, competitive inhibition by benzamidine and the formation of a stable p-guanidinobenzoyl acyl enzyme intermediate. Inactivation by affinity labeling with Z-LysCH 2Cl was successful in contrast to the inertness of Tos-LysCH 2Cl.

Antineuraminidase Antibody Response of Man to Influenza Virus Neuraminidase N2: Results Obtained with an Improved Hemagglutination Inhibition Technique and an Enzyme Inhibition Test

Abstract Sets of sera obtained from subjects vaccinated or infected with influenza A2 strains from 1957 to 1968 were examined by an improved hemagglutination inhibition procedure for measuring antineuraminidase antibodies with recombinant viruses, and by a neuraminidase inhibition test with human serum Cohn fraction IV-4 as substrate. Recombinants prepared with viruses containing 1957 and 1968 antigenic variants of neuraminidase were employed. The improved HI procedure was found to be as reliable as the NI test for detecting antineuraminidase antibody and antineuraminidase antibody increase. Antineuraminidase antibody was generally not found on first exposure to A2 strains but antibody increase occurred with increasing frequency during subsequent years. The antineuraminidase antibody response of man is broad, making recognition of antigenic enzymatic variants difficult by use of human serum.

Inhibition of collagen-induced platelet aggregation by normal plasma

Normal plasma has been found to inhibit the platelet aggregation-inducing effect of collagen in a time consuming reaction independent of temperature. Collagen treated with serum and washed has reduced reactivity which can be restored to normal by treatment with 1.5 M sodium chloride. On the basis of this result, it is suggested that inhibition results from adsorption to collagen of a plasma component. The inhibitory plasma component is destroyed at 56 degrees C, is unstable below pH 7, and migrates with the alpha globulins on starch block electrophoresis at pH 8.6. On the basis of ultrafiltration and sucrose density gradient ultracentrifugation studies, a molecular weight in the range of 330,000 is suggested and there may be an additional component of considerably greater size. Partial purification can be achieved by ion exchange chromatography. The purified fraction was completely inactivated by incubation with trypsin. Partially purified fractions inhibit cationic platelet aggregators such as collagen, polylysine, and hexadimethrine but do not affect anionic aggregators such as succinylated collagen and sodium stearate. Normal plasma and serum inhibit succinylated collagen and stearate. Stearate is inhibited by crystalline albumin and Cohn fraction IV-4. It is suggested that plasma proteins may regulate platelet adhesion to collagen and other vessel wall materials.

Enzymes Produced by a Pseudomonas Species Which Inactivate Inhibitors of Certain Viral Hemagglutinins

Abstract Elastase and phospholipase C which were purified from cultures of a psychrophylic Pseudomonas sp. were examined for ability to inactivate certain viral hemagglutinin inhibitors present in human sera. Both enzymes were required for inactivation of enterovirus and reovirus inhibitors, but adenovirus inhibitors were inactivated by the elastase alone. The Pseudomonas sp. elastase had much greater inhibitor-destroying activity than did pancreatic elastase or Flavobacterium elastase; the activity of the Pseudomonas sp. phospholipase C was comparable to that of phospholipase C from Clostridium perfringens. Cohn fraction III-0 (containing predominantly β lipoprotein) and fraction IV-1 (containing predominantly α lipoprotein) both inhibited enterovirus hemagglutinins. Immunoelectrophoretic studies showed that the combination of Pseudomonas elastase and phospholipase C produced marked alterations in both the α1 and β lipoproteins in human sera. Kaolin absorption also removed both α1 and β lipoproteins. Both of these treatments inactivate enterovirus HA inhibitors. Treatment of sera with heparin-MnCl2 or dextran sulfate-CaCl2, which precipitate β lipoproteins, removed only a portion of the inhibitory activity for enterovirus hemagglutinins. These findings suggest that enterovirus hemagglutinin inhibitors are present in both α and β lipoproteins of human sera.