Carboxymethyl Cellulose Column Chromatography Research Articles

IgE reactivity to type I collagen and its subunits from tilapia (Tilapia zillii)

Acid-soluble collagen (ASC) was isolated from the skin of tilapia (Tilapia zillii) via acetic acid (HAc) extraction and NaCl precipitation. ASC from tilapia consists of α chains (α1 and α2), β chains and γ chains and is classified as type I collagen. A comparison of the properties of tilapia collagen and silver carp parvalbumin showed that tilapia collagen was less stable under heat treatment and more resistant to pepsin digestion. Both tilapia collagen and silver carp parvalbumin were degraded at pH 2.0 but stable at pH 3.0–11.0. Subunits α1 and α2 were further purified from tilapia collagen by carboxymethyl (CM) cellulose column chromatography with linear gradient elution and stepwise elution, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoblotting results demonstrated the specific IgE activity of different fish-allergenic patients’ sera towards the α1 and α2 chains of tilapia collagen. It can be inferred that tilapia collagen and its subunits are all potential allergens.

Characterization of hydrogen peroxide removal activities in mouse hemolysates: catalase activity and hydrogen peroxide removal activity by hemoglobin

Hydrogen peroxide removal activities in normal and acatalasemic mouse hemolysates were examined to determine the optimal temperature of catalase. From thermal stability of the removal activities in hemolysates, the removal activities were divided into two activities. The removal activity deactivated at lower temperature was catalase, and the 50% inactivation was observed after 10 min incubation at 47.2±0.5°C for normal hemolysates and 34.0±0.8°C for acatalasemic ones. The removal activity deactivated at a higher temperature remained after the addition of sodium azide, and the 50% inactivation was observed at 63.5±1.4°C. After separation of the removal activities by carboxymethyl-cellulose column chromatography, the removal activity deactivated at higher temperature was attributed to the activity by hemoglobin. From Lineweaver-Burk plot analysis of the removal rates by hemoglobin at 37°C, the Michaelis constant for hydrogen peroxide and the maximum velocity were 201±53 μM and 5.37±1.39 μmol/s per g of Hb, respectively. Removal rates by hemoglobin in mouse hemolysates at 37°C in 70 μM hydrogen peroxide were 1.32±0.12 μmol/s per g of Hb. Catalase activity (k/g Hb: rate constant related to the hemoglobin content) in normal mouse hemolysates was 104±12 at 25°C and 117±10 at 37°C, and that in acatalasemic hemolysates was 10.5±1.7 at 25°C. These results indicate that activity of hydrogen peroxide removal by hemoglobin is substantial and the activity in acatalasemic hemolysates is predominant at low concentration of hydrogen peroxide.

Heavy cigarette smokers show higher mutagenicity in urine

We examined the mutagenicity of cigarette smoker's urine in 32 healthy male cigarette smoker and 37 healthy male non-smoker. Twenty-four-hour urine specimens were subjected to blue rayon extraction which selectively adsorb polycyclic compounds, after which the elutions were fractionated by carboxymethyl cellulose column chromatography for removing antimutagenic compounds. The mutagens were measured by using an S9-mediated Salmonella mutagenicity test on strain TA98. Compared with those with non-smokers, smokers' urine showed a significantly higher urinary level of mutagens in the acid-elutable and in the sum of all chromatography fractions. A similar tendency was also seen in the alkali-elutable fraction. The subjects were classified into three groups according to the number of smoked cigarettes. Heavy smokers, who smoked more than 20 cigarettes per day, showed a significantly higher urinary level of mutagens than both non-smokers and light smokers especially in the acid-elutable and in the sum of all chromatography fractions. Our findings suggest that smokers are exposed to a great amount of polycyclic carcinogens and mutagens by cigarette smoking. These results also suggest that urinary level of mutagens measured by using blue rayon extraction combined with carboxymethyl cellulose chromatography could be a good index for estimating the exposure to carcinogens and mutagens such as polycyclic compounds.

Urinary Mutagens and Lifestyle Factors

Background.Lifestyle determines the amount of exposure to environmental carcinogens/mutagens. We examined the relationship between various lifestyle factors and the urinary level of mutagens, which reflects both exposure dose and metabolism of these carcinogens/mutagens.Methods.Twenty-four-hour urine specimens obtained from 69 males were subjected to blue rayon extraction, after which the elutions were fractionated by carboxymethyl cellulose column chromatography. The mutagens were measured by using an S9-mediatedSalmonellamutagenicity test. The subjects were classified into three groups according to their total scores on a questionnaire regarding eight health practices: cigarette smoking, alcohol drinking, nutritional balance, eating breakfast, sleeping hours, working hours, physical exercise, and mental stress.Results.Compared with those with “poor” health practice, subjects with “good” health practice showed a significantly lower urinary level of mutagens in all chromatography fractions, as well as in the acid- and alkali-elutable fractions. Cigarette smoking and nutritional balance were the most highly correlated factors.Conclusions.Our findings show that poor health practices increase the urinary level of mutagens, suggesting that a healthy lifestyle reduces exposure to carcinogens/mutagens and may reduce cancer risk.

Determination of Covalently Bound Hypusine and Deoxyhypusine to Protein Using Submilligram of Protein Samples by HPLC.

A sensitive and reliable method for the determination of hypusine and deoxyhypusine in eIF-5A protein, an initiation factor of protein synthesis, was developed. An advantage of this method is the use of N epsilon-(5-aminopentyl)lysine, an analogue of deoxyhypusine, as an internal standard. The application made it possible to determine hypusine in less than a mg of protein samples from cultured HTC cells and rat organs. After acid hydrolysis of protein samples to which had been added the internal standard, the hydrolysates were fractionated by carboxymethyl cellulose column chromatography. Also, diamine fractions containing a few pmol of hypusine and deoxyhypusine were successfully analyzed by a reversed phase HPLC with a fluorescence detection of o-phthalaldehyde. The method was applied for the determination of hypusine and deoxyhypusine in drug-treated HTC cells and normal rat organs. The results from HTC cells were discussed based on the known effects of each drug on hypusine biosynthesis.

A simple method for separation of the protein crystal from Bacillus thuringiensis using carboxymethyl cellulose column chromatography

A simple and a rapid technique to separate intact crystals from spores and cell debris of Bacillus thuringiensis using a carboxymethyl cellulose (CM-cellulose) column is described. B. thuringiensis subsp. kurstaki, a toxic strain to lepidopteran insects that produces small bipyramidal crystals, and B. thuringiensis subsp. yunnanensis, a nontoxic strain producing unusually large bipyramidal and spindle-shaped crystals were used as test organisms. Spores and cell debris were washed in sodium acetate and sodium phosphate buffers and the crystals bound to the CM-cellulose were eluted with Tris-EDTA buffer, regardless of their size and shape. Light microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purity of the crystal was > 98%. The percent recoveries of crystals from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. yunnanensis were approximately 14 and 25, respectively.

Some biochemical characteristics of a partially purified extracellular keratinase from trichophyton schoenleinii

A Trichophyton schoenleinii (T. schoenleinii) strain from tinea favus was cultured in a liquid medium, from which an extracellular keratinase extract was obtained. The keratinase was partially purified with carboxymethyl-cellulose (CMC) column chromatography. Some biochemical characteristics of the keratinase were then examined. Its molecular weight was estimated to be 38,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature was 50 degrees C, and the optimal pH value was 5.5. The keratinolytic activity was specifically increased by Fe++ and Fe .

Prenatal Diagnosis of Hemoglobinopathies: A Potential Application of High Performance Liquid Chromatography

High performance liquid chromatography was compared with carboxymethyl cellulose column chromatography data for analysis of globin chain composition and biosynthetic ratios in fetal blood. A formula was derived for mathematical correction for losses of small amounts of globin protein on the high performance liquid chromatography column. Radioactive globin chain ratios by high performance liquid chromatography are equivalent to those by carboxymethyl cellulose column chromatography, but only when carrier Hb A is added. We conclude that high performance liquid chromatography provides a suitable alternative to carboxymethyl cellulose columns, and has several advantages.

A procedure for extracting staphylococcal enterotoxin from foods at elevated temperatures and low centrifugal forces

Various low relative centrifugal forces (RCFs) were tested for their effectiveness in ‘cleaning up’ extracts of staphylococcal enterotoxin from food homogenates. RCFs of at least 10 000 × g were effective if centrifugations were for 30 min periods or longer. Below 8000 × g, and down to about 1000 × g, the cleaning up of extracts was effective only if centrifugation was preceded by filtration of food homogenates. Investigations of the thermal stability of enterotoxin during cleaning up of extract at higher than refrigeration temperatures showed that centrifugation temperatures of up to 40°C had little adverse effect on the serological activity of the toxin. Based on these findings a procedure was devised for extracting enterotoxin from foods using basic bench top centrifuge at ambient temperatures. Separation of enterotoxin from food proteins in the extract was achieved by carboxymethyl cellulose-column chromatography. Levels of enterotoxin A detectable by the microslide gel double diffusion method following extraction by this procedure were 2.5 ng/ml for milk, 2.5 ng/g for yogurt and 5 ng/g for cheese and corned beef.

Amino-acid sequences of the two major components of adult hemoglobins from the stump-tail monkey, Macaca speciosa.

The adult Stump-Tail Monkey (Macaca speciosa) was found to have two major hemoglobin components (Hb 1 and Hb 2) which were separated by carboxymethyl cellulose column chromatography. The tryptic peptides of the alpha and beta chains from the two components were isolated and sequenced. The peptides were aligned based on the homology of their sequences with that of human adult hemoglobin. Only one amino-acid difference was found between the alpha chains from Hb 1 and Hb 2 at the 15th position from the N-terminus. On the other hand, the beta chains from the two hemoglobin components were considered to be identical.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

Studies of Bitter Peptides from Casein Hydrolyzate. I. Synthesis of Bitter Peptide BPIa Corresponding to Arg–Gly–Pro–Pro–Phe–Ile–Val from Casein Hydrolyzate by Alkaline Proteinase of Bacillus subtilis

Abstract A bitter heptapeptide BPIa was synthesized and compared with the natural peptide, isolated by Minamiura et al. from casein hydrolyzate, by means of thin layer chromatography, paper electrophoresis, and carboxymethylcellulose column chromatography. All results for the two peptides matched very closely each other. The synthetic BPIa has an extremely bitter taste with its threshold value 0.05 mM. It is one of the most bitter compounds such as phenylthiourea and quinine.

Characterization of mutagenic fractions in beef extract and in cooked ground beef. Use of blue-cotton for efficient extraction.

Mutagenic components in commercial beef extract and in cooked ground beef were adsorbed from their aqueous solutions on cotton bearing covalently linked trisulfo-copper-phthalocyanine residues (blue-cotton). By repeating the adsorption and elution, efficient concentration of the mutagenic components with a satisfactory overall recovery was achieved. Carboxymethyl cellulose column chromatography was found to be an excellent means to separate 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), two strong mutagens that have previously been found in heated beef samples. Chromatography of the mutagenic components of beef extract on this column gave two mutagenic fractions which corresponded to MeIQx and IQ in elution profile. In reversed-phase high performance liquid chromatography, the major active component of the MeIQx fraction and that of the IQ fraction behaved identically with standard samples of MeIQx and IQ, respectively. The contents of these mutagens in a sample of Difco beef extract were estimated at 200-300 ng of MeIQx and 20-40 ng of IQ per gram. By the same fractionation procedures, mutagenic substances in the cooked beef were fractionated into MeIQx-type and IQ-type components. The activity distribution among these two fractions was similar to that found for beef extract.

Structural properties of Type I collagen isolated from chickens with scoliosis.

This study examines biochemically the Type I collagen isolated from skin of chickens that develop idiopathic scoliosis. Previous studies indicate a defect in collagen exists in these chickens. Alpha 1 (I) and alpha 2 chains were separated by gel filtration and carboxymethyl cellulose column chromatography and were then subjected to the analytical techniques of sodium dodecyl sulfate gel electrophoresis, Staphylococcus aureus V8 protease digestion, cyanogen bromide peptide mapping and amino acid analyses. In all categories, the scoliotic alpha 1 (I) and alpha 2 chains were identical to alpha chains isolated from normal chickens. These data suggest that the altered properties of collagen solubility and connective tissue stress relaxation seen in these scoliotic chickens are not a manifestation of an altered primary structure of the alpha chains or post-translational modification affecting chromatographic elution profiles or electrophoretic migration patterns.

6] Arachidonic acid-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes

This chapter presents the procedure for purification and assay of arachidonic acid-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. All operations in purification were done at 0–4 °, crude extraction cells were sonicated 15 times for 30 sec in 60 ml of 50mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Further procedure steps are (1) acetone fractionation, (2) carboxymethyl-cellulose column chromatography, (3) gel filtration on sephadex G-150, and (4) hydroxyapatite column chromatography. The enzyme was eluted at approximately 350 mM buffer concentration. The active fractions were pooled and concentrated by ultrafiltration to about 1 ml. The activity of arachidonic acid-15-1ipoxygenase was assayed by measuring the sum of the radioactivities converted from [1-14C]arachidonic acid to 15-hydroperoxyeicosatetraenoic and its decomposition products. The radioactivity was determined in a scintillation counter. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the formation of 1μmol of product in 2 min at 30 °.

Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly.

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high-speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.

Hemoglobin Petah Tikva (a 110 Ala → Asp): A New Unstable Variant With α-Thalassemia-Like Expression

AbstractHemoglobin Petah Tikva, a new unstable α-Chain structural variant, was identified in two unrelated children whose families are from the Iraqi Jewish community of Baghdad. Both children have α-thalassemia. with hematologic features of mild HbH disease. The abnormal hemoglobin from the two patients did not separate from hemoglobin A by electrophoresis or chromatography, but the mutant α-chain emerged ahead of αA in carboxymethylcellulose column chromatography, allowing it to be isolated in pure form. Hemoglobin Petah Tikva was unstable in solution and could also be isolated by isopropanol precipitation. Structural analyses indicate that the variant α-Chains contain a normal number of amino acids and have a substitution of Ala → Asp at α 110. Each of the patients with HbH disease has one parent with hematologic findings and globin chain synthesis ratios that are characteristic of α-thalassemia trait, and a hematologically normal parent who is a carrier of the variant α-chain of Hb Petah Tikva. The patients each appear to be doubly heterozygous for α-thal-1 and for the structurally abnormal hemoglobin. The mutant α-chains of Hb Petah Tikva were synthesized by reticulocytes of the heterozygous carriers, but only traces of αPT globin could be detected in their blood, suggesting that the abnormal α-chains underwent postsynthetic degradation or precipitation in these individuals. In the patients with Hb Petah Tikva in combination with α-thalassemia, the normal and mutant α-chains were synthesized by bone marrow erythroid cells at rates proportional to their concentrations in the blood, but there was a substantially decreased rate of synthesis of the variant α-chain by their blood reticulocytes. The α-thalassemia-like expression of Hb Petah Tikva appears to result from instability of the abnormal α-chain as well as from the premature termination of its synthesis during erythroid cell maturation.

Biochemical characterization of a toxin from indian cobra ( Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra ( Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld 50, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.

Isolation and properties of somatic and testicular cytochromes c from rat tissues

Somatic ( c s ) and a testis-specific ( c t I) cytochromes c were purified to homogeneity from rat tissues (heart, liver, kidney, and testis). The purification procedure involved (1) homogenization of tissues at pH 4.5, (2) treatment with methanol-chloroform solvents, (3) hydroxylapatite column chromatography, (4) carboxymethyl-cellulose column chromatography, and (5) Sephacryl S-200 gel filtration. The isolated cytochromes c were free from polymeric and other “modified” forms, and did not bind CO, azide, or cyanide. The absorption maxima and the molecular weights of both cytochromes c s and c t I were identical. The ratio of A 549.5 nm ( reduced) A 280 nm ( oxidized) for cytochromes c s averaged 1.28. The unique properties of cytochrome c t I, compared to somatic cytochrome c, were as follows: (1) different elution profiles from hydroxylapatite and carboxymethyl-cellulose column chromatography experiments, (2) less basic intrinsic molecular charge shown by the slow mobility in native polyacrylamide gel electrophoresis, (3) probable asymmetric molecular shape as evidenced from gel filtration experiments, (4) significantly higher millimolar extinction coefficient values (33.6 at 549.5 nm), (5) a low ratio (1.04) of A 549.5 nm ( reduced) A 280 nm ( oxidized) , and (6) difference of about 20 amino acid residues per mole.