Thin-layer Gel Filtration Research Articles

Preparation of radiolabeled clonidine for use in radioimmunoassay

The preparation of stable and immunoreactive radioiodinated clonidine for the development of a radioimmunoassay of clonidine is described. Tyrosylmethyl ester (TME) conjugate of carboxylic acid derivative of clonidine was radioiodinated using chloramine T as the oxidizing agent. The radioiodinated clonidine was purified by thin layer chromatography, gel filtration and solvent extraction. Radiochemical purity, specific activity and immunoreactivity were determined. The stability of the labeled compound during storage was tested in various solvents at different temperatures.

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.

Calcium-induced aggregation of the urea-deaggregated human lens insoluble protein

Calcium-induced aggregation of the urea-deaggregated insoluble protein (US-fraction) derived from human lens was investigated by use of a simple light scattering method. The initial rate of aggregation was shown to increase with progress of senescence of the lens, and to be much higher in the nucleus as compared to the cortex. The aggregation was found to be more active in the US-fraction from cataractous lens than in that of normal lens at same age. A calcium sensitive polypeptide (CSP) was isolated from the US-fraction from cataractous human lenses by gel filtration followed by DEAE-cellulose column chromatography in the presence of 7 m urea. The molecular size of the CSP in the dissociated state was assessed to be 4·2 kDa by urea-SDS-PAGE, and the size in the associated state to be 450 kDa by thin layer gel filtration. With addition of calcium ion to the latter, it aggregates rapidly to form much larger aggregates and eventually precipitates out. The aggregation rate of the CSP is shown to increase exponentially with an increase in the concentration of both CSP and calcium ion, indicating the involvement of complicated mechanisms in the aggregation process. Effects of divalent ions other than calcium ion on the aggregation process were also examined. It is revealed that the effect of manganese ion is little less than that of calcium ion, whereas magnesium and strontium ions are much less effective as compared to the former two ions.

Some characteristics of aggregates of IgG and plasma proteins in heat-treated factor VIII concentrates

Eight batches of commercial heat-treated and one untreated factor VIII concentrate from 6 producers were analyzed for their content of IgG, IgG subclasses, IgG aggregates and the presence of other plasma proteins combined with the IgG as well as for anticomplement activity. Methods used were thin-layer gel filtration, immuno-gel filtration, spot immuno-precipitate assay in a double antibody version and an agarose plate haemolysis inhibition assay of complement fixation. The IgG content varied from 0.1-6.90 g/l. In all preparations IgG existed as monomers and aggregates. Associated with the IgG were also found, at significantly increased amounts compared to normal serum and intravenous immunoglobulin, one to four of the following plasma proteins; fibronectin, fibrinogen, von Willebrand factor antigen, Clq, albumin and IgA. Three batches from two producers had high anticomplementary activity, presumably caused by the IgG aggregates. Two of these deviated strikingly from normal human serum pools in percent distribution of IgG subclasses. It is hypothesized that these aggregates can induce side effects or cause immunological aberrations.

A STUDY OF ADENOSINE DEAMINASES IN HUMAN SERA: 114

Two different molecular forms of adenosine deaminase have been found in human tissues: the high molecular weight enzyme and the low molecular weight enzyme, designated as the A form and the C form respectively. These two enzyme forms are present in various proportions in different tissues. In previous studies, the two enzyme forms were separated and identified by gel filtration column chromatography and spectrophotometric measurements of enzyme activity in the elution fractions. A rapid and efficient method is needed to perform routine analysis of the enzyme form distribution, especially in samples containing little enzyme activity, and to study the interrelationship of the two enzyme forms. This study shows the development of the thin-layer gel filtration technique for the separation and the identification of the two molecular forms of adenosine deaminase in human tissue extracts. Possible clinical application of this technique and the enzyme system is discussed. This work was supported by a grant from the Indiana Delaware County Cancer Society.

Chromatographic and biological comparison of99mTc-DMSA prepared by direct labelling and ligand exchange reaction

99mTc-dimercaptosuccinic acid /DMSA/ is one of the most favourable agents used for renal scintigraphy. This radiopharmaceutical was prepared in two different ways: by direct labelling of tin/II/-dimercaptosuccinic acid and by ligand exchange reaction from99mTc-gluconate at tracer concentration of technetium under acidic condition. The complexes formed were compared using paper chromatography, thin layer chromatography, gel filtration and electrophoresis. Their biodistribution was studied on rats.

Thin layer gel filtration immunofixation: Identification of abnormal molecular weight immunoglobulins or related fragments

A simple procedure is described to identify immunoglobulin (Ig) fragments without isolation from biological fluids. It involves an initial separation based on molecular weight (MW) by thin layer gel filtration (TLG) followed by immunofixation (IF) in cellulose acetate strips with monovalent antisera. TLG-IF permits detection of differences about 10,000 Da MW and specific immunological typing, making it a useful tool in the accurate identification of protein fragments.

A low-molecular-weight cadmium-binding substance in human and rat livers and human blood

A cadmium-binding substance with a molecular weight even lower than that of metallothionein was demonstrated on Sephadex G-75 gel filtration chromatography of the soluble fractions from newborn human and adult rat liver homogenates and adult human hemolysate which were mixed with CdCl 2 in vitro. This substance was purified from rat liver extracts by gel filtration and ion-exchange column chromatography and characterized by 6 m guanidine · HCl thin-layer gel filtration chromatography and N-terminal and total amino acid analyses. The results showed that the isolated low-molecular-weight cadmium-binding substance was a cadmium-reduced glutathione complex, whose molecular weight was found to be approximately 1400 by Sephadex G-15 gel filtration.

Charge and molecular weight heterogeneity of EDTA-extractable proteins from calf lens membranes

The EDTA-extractable proteins (EEP) of calf lens fiber cell membranes have been further characterized. Fiber EEP has been purified by gel filtration and resolved into eight bands with molecular weights of 30–38 Kdalton by SDS-polyacrylamide gel electrophoresis. For epithelial EEP the same range has been obtained. In agreement with these findings a value of 33 K has been determined for fiber EEP by Sephadex G200 thin-layer gel filtration, while 34 K dalton was found by high-performance gel permeation chromatography in combination with low-angle laser light scattering (HPGPC-LALLS). The isoelectric focusing patterns of fiber and epithelial EEP show considerable charge heterogeneity. By two-dimensional electrophoresis the relation molecular weight-isoelectric point has been established for most EEP components. Peptide maps of the individual protein bands of fiber EEP differ from each other and from those of the βB p-, βB 1a- and βB 1b-crystallin bands, which have about the same molecular weight. From our results we conclude that EEP is not an oligomeric nor a multisubunit protein, but a collection of different extrinsic membrane proteins, biochemically unrelated to lens crystallins. The fact that removal of the cytoskeleton by urea-treatment of the membranes is a prerequisite for its isolation by EDTA or EGTA suggests that EEP is bound to the inner surface of the plasma membranes, probably via calcium.

Structural investigation of radiation-induced aggregates of ribonuclease.

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.

Creatine kinase from the electric organ of Electrophorus electricus (L.)—isozyme analysis

1. 1. Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract. 2. 2. Upon acetone fractionation of the aqueous extract of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity. 3. 3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure. 4. 4. The implications of this finding with the muscular origin of the electric organ are discussed.

Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver.

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

Phenobarbital-digitoxin interaction in the guinea pig liver.

The influence of phenobarbital pretreatment on liver concentration of digitoxin and its metabolites was studied in guinea-pigs after i.p. administration of the cardiac glycoside. During the first hour an increase in liver uptake was observed in pretreated animals. The differences detected in the hepatic subcellular distribution do not seem to explain the higher concentrations found in the liver of phenobarbital pretreated animals. About 80% of the liver radioactivity was found in the supernatants. Inhibition of digitoxin biotransformation by phenobarbital was demonstrated by chromatographic analysis of the organic soluble compounds present in the supernatants. The possible binding of digitoxin and its metabolites to soluble proteins of liver cytosol was excluded by thin-layer gel filtration. The decrease in digitoxin biotransformation seems to be the reason for the increase in liver uptake and for the decrease in bile concentrations, observed in phenobarbital pretreated animals.

Determination of the molecular weight of alpha-amylase, as the enzyme-inhibitor complex, using thin layer gel filtration on Sephadex.

Thin layer gel filtration on Sephadex was performed as a simple method for recognition of the small molecular weight differences of human alpha-amylases from different sources. Activity was located with dry chromogenic substrate, using a replica technique. Undesirable interaction between the gel matrix and substrate binding sites on the enzyme, which causes an anomalous decrease in the migration rate of the enzyme protein, was suppressed by preincubation of the enzyme with appropriate inhibitor. Gradual masking of substrate binding sites of the enzyme by increasing concentrations of amylase inhibitors resulted in two distinct migration rates for the enzyme in thin layer gel filtration. This suggests the existence of two substrate binding sites in the enzyme molecule. Together with thin layer gel affinity chromatography on a mixture of Sephadex and ConA-Sepharose, the method yielded useful data on the molecular weight of amylase and its glycosylated forms and served as a valuable tool for the differential diagnosis of macroamylasaemia.

Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein.

Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2. We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer. The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography. The A2 protein's identity was confirmed by two-dimensional gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide. Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide. The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer.

NADP-specific isocitrate dehydrogenase from Escherichia coli V. Multiple forms of the enzyme

Two forms of NADP-specific isocitrate dehydrogenase ( threo-D S-isocitrate: NADP + oxidoreductase (decarboxylating), EC 1.1.1.42) in Escherichia coli have been resolved by polyacrylamide gel isoelectric focusing and electrophoresis. Incubation of the enzyme with Mn 2+ plus isocitrate prior to focusing resulted in the formation of an additional form of the enzyme, presumably the enzyme-manganese-isocitrate complex. Glycerol, a cryoprotectant used to stabilize the enzyme during purification and storage, also stabilized it during focusing, but was not necessary during electrophoresis. Thin-layer gel filtration did not reveal any differences in molecular weight between the different species of isocitrate dehydrogenase.

Fatty acid binding proteins in the serum of various animals

1. 1. A technique is described to study the binding of fatty acids to albumin in serum by use of thin-layer gel filtration. 2. 2. The presence of a protein which binds [ 14C]oleate is established in higher vertebrates, teleosts and a petromyzone. 3. 3. No fatty acid binding protein was observed in the six elasmobranchs studied and the results with crustaceans were ambiguous. 4. 4. The aspects of functional evolution of serum albumin are discussed.

Liaison spécifique du cortisol aux protéines plasmatiques chez le Cobaye, de la naissance au sevrage

We have estimated two parameters of guinea-pig adrenal function during the neonatal period: --the concentration of plasma cortisol by competitive binding to protein; --the association constant, Ka, and total capacity of CBG binding (NS) by the thin-layer gel filtration technique. The concentration of plasma cortisol, very high at birth, declined markedly between neonatal days 1 and 10 and then remained constant between days 10 and 20, the time of weaning. Ka was 0.25.10(7).M-1 in the newborn and the adult of both sexes. This value was lower than that of other species. NS, which was very high at birth (111 +/- 5 micrograms/100 ml in the male; 101 +/- 5 micrograms/100 ml in the female), decreased rapidly during the first two neonatal days. At that time, it reached adult levels in the female (45 +/- 5 micrograms/100 ml) and remained stable. Adult binding capacity (16 +/- 2 micrograms/100 ml) was reached in the male after neonatal day 20.

Estradiol binding in rat thymus cells.

Estradiol binding in preparations of female rat thymi is demonstrated by three different approaches (dextran-coated charcoal method, immunofluorescence technique and thin-layer gel filtration). Scatchard's analysis of [3H]-estradiol proves consistent with the existence of a single class of receptor sites, having a dissociation constant (Kd) of about 2 x 10(-10) M. Steroid binding specificity in the thymus is similar to that found in the uterus. On thin-layer gel filtration the binding appears in the 8S region similar to that observed for the uterus. With the immunofluorescence sandwich technique, estradiol proves to bind to a specific cytoplasmic component that subsequently migrates into the nucleus.

Lens membranes VI. Some characteristics of the EDTA-extractable protein (EEP) from bovine lens fiber membranes

After urea-treatment of calf lens fiber membranes, a well defined extrinsic membrane protein fraction (EEP) can be extracted by EDTA-solution. It contains polypeptides of mol. wt. 32K and 35K (SDS-gel electrophoresis). Thin-layer gel filtration shows that without detergent no oligomers are formed. It is easily lost by its adsorption characteristics, however, it can be purified from contaminating α crystallin by gel filtration. The 35K fraction comprises components with isoelectric points at 4·6–4·7 (double band) and between 6·2 and 7·2. The 32K fraction shows components at 4·6–4·7 and 6·2 as was found by two-dimensional gel electrophoresis. The method of isolation, the isoelectric focusing pattern, and the amino acid and mol. wt. composition distinguish EEP clearly from the soluble crystallins. EEP comprises two distinct antigenic components with isoelectric points between 6·2 and 7·2. This was revealed by immunoelectrofocusing of EEP vs. anti-EEP antiserum from rabbit. Combining the results of two-dimensional electrophoresis and immunoelectrofocusing it was found that the 32K and 35K polypeptide have different antigenic properties. Traces of crystallins are difficult to remove. However, by means of specific anti-crystallin antisera it has been shown that EEP has a determinant in common with γ crystallin.