Isolation and Amino Acid Sequences of Small Tryptic Peptides from the Oxidized Ala Chain of Ricin D

Twelve tryptic peptides as well as free arginine were isolated from the performic acid-oxidized Ala chain of ricin D by gel filtration on Sephadex G–25 and Dowex 1×2 column chromatography followed by paper chromatography. Total number of the amino acid residues in these peptides accounted for 90 out of 263 residues in the Ala chain of ricin D.

The amino acid sequences of nine peptides were determined by manual Edman degradation.     

Purification of ricin A1, A2, and B chains and characterization of their toxicity

This paper describes a protocol for the preparation of highly purified A (A1 and A2) and B chains of the plant toxin, ricin, and biochemical and biological characterization of these proteins. Intact ricin was bound to acid-treated Sepharose 4B and was split on the column into A and B chains with 2-mercaptoethanol. The A chains were eluted with borate buffer containing 2-mercaptoethanol. A1 and A2 were then partially separated by cation exchange chromatography and the contaminating B chain was removed by affinity chromatography on Sepharose-asialofetuin and Sepharose-monoclonal anti-B chain. The B chain was eluted from the Sepharose 4B column by treatment with galactose and was further purified by cation and anion exchange chromatography; contaminating A chains were removed by affinity chromatography on Sepharose-monoclonal anti-A chain. The purified A and B chains were active as determined by their ability to inhibit protein synthesis in a cell-free assay and their binding to asialofetuin, respectively. Furthermore, by polyacrylamide gel electrophoresis, toxicity in mice, and toxicity on several different cell types, both A and B chains were shown to be minimally cross-contaminated. Finally, it was shown that ammonium chloride significantly enhanced the nonspecific toxicity of B chains for cells in vitro. In contrast, ammonium chloride did not enhance either the nonspecific toxicity of A chains in vitro or the specific toxicity of A chain-containing immunotoxins prepared with the highly purified A1, A2 chains.     

Subunit Structure of Castor Bean Hemagglutinin

Two constituent polypeptide chains of castor bean hemagglutinin (CBH-A) were isolated from the performic acid-oxidized or reduced-carboxymethylated CBH-A by chromatography on DEAE-cellulose or Sepharose 4B. From the analyses of the N-terminal amino acids, the amino acid compositions and the tryptic peptides of each chain, it was found that the larger chain with mol. wt. 34,000 and the smaller chain with mol. wt. 31,000 were homologous with the Ala and He chains of ricin D, respectively, and the subunit structure of CBH-A is represented as (α′/β′)₂ in relation to αβ of ricin D.     

Effects of Physical and Chemical Treatments on the Biological Activity of Ricin D

Effects of physical and chemical treatments on the cytoagglutinating activity, toxicity and inhibitory activity of cell-free protein synthesis of ricin D or its constituent polypeptide chains were investigated. The results indicated that the isolated polypeptide chains were much less stable than intact ricin D in acidic pH, heating as well as chemicals, and the Ala chain was more unstable than the lie chain.

Chemical modifications of ricin D with specific reagents revealed that the tryptophan and tyrosine residues as well as the carboxyl groups participated in the phenomena of cyto- agglutination and toxic action of ricin D, whereas arginine residues were considered not to be directly involved. Trinitrophenylation of free amino groups did not result in a loss of cytoagglutinating activity, whereas caused a loss of toxicity, suggesting that free amino groups in the lie chain were involved in the toxic action of ricin D.     

Isolation of Tryptic Peptides from the lie Chain of Ricin D and the Sequences of Some Tryptic Peptides Including N- and C-Terminal Peptides

Twenty tryptic peptides were isolated from the performic acid-oxidized He chain of ricin D by Dowex 1 × 2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the He chain of ricin D were established to be NH₂–Ile–Phe–Pro–Lys–Gln–Tyr–Pro–Ile–Ile– and Cys–Ala–Pro–Pro–Pro–Ser–Ser–Gln–Phe, respectively.     

Primary structure of human class II histocompatibility antigens (HLA-D). I. Isolation, purification and characterization of the HLA-D alpha/beta chain complex from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3;B7,7;Dw2, 2;DR2,2;MT1,1;DC1,1;MB1,1

The complex of alpha and beta chains of HLA-D membrane antigens has been isolated from a lymphoblastoid homozygous B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1), by an exclusively chemical two-step procedure and characterized by electrophoresis as well as isoelectric focusing in polyacrylamide gel. Cells were gained using long term cultivation in large scale, the crude membrane by differential centrifugation. The proteins of the crude membrane were then solubilized in NP-40, pH 5.0. The first purification step for HLA-D antigens consisted in an ion-exchange chromatography on carboxymethyl cellulose using the solubilization buffer. By this procedure the complex of proteins with relative molecular masses of Mr approximately 34 000 and Mr approximately 29 000 was in a high percentage not bound to the carboxymethyl cellulose. The bound fraction contained the HLA-A, -B and -C antigens and a component with Mr approximately 31 000 corresponding to the well known Ii-fraction. The bound proteins could be recovered from the column by a sodium chloride gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS buffer, pH 7.2 and then submitted to the second purification step, the Sephacryl S-300 chromatography. By this procedure the corresponding complex could be further separated from higher and lower molecular proteins. The complex was used as the starting material for the separation of alpha and beta chains. Amino-acid sequences established of the isolated chains have already been communicated.     

The Mass Spectra of Cotylenol and Cotylenins

A toxic protein, distinct from ricin D, was isolated from castor beans produced in Japan and referred to as ricin E. Ricin E was extracted from defatted meal of castor beans and purified by gel filtration through Sephadex G-75 column followed by DEAE- and CM-cellulose column chromatographies. Ricin E was found to be a glycoprotein and had two N-terminal amino acids, Ile and Ala, which were identical to those of ricin D. The molecular weight of ricin E was 64,000 by SDS-polyacrylamide gel electrophoresis and its sedimentation coefficient was 4.45 S. The isoelectric point of ricin E, estimated to be 8.8 by ampholine electrophoresis, was higher than that of ricin D. Ricin E had equal toxicity for mice and equal cytoagglutinating activity for Sarcoma 180 ascites tumor cells to those of ricin D, and this cytoagglutination was inhibited by galactose.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

Rat haemoglobin heterogeneity. Two structurally distinct alpha chains and functional behaviour of selected components.

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two alpha chains and three non-alpha chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor alpha chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.     

Structure and toxicity of pure ricinus agglutinin

Highly purified ricinus agglutinin was found to inhibit protein synthesis in HeLa cells. This effect could be prevented by the addition of the specific antiricinus agglutinin serum, whereas specific anti-ricin serum did not protect the cells, demonstrating that the toxic effect of ricinus agglutinin is not due to contamination with ricin.After reduction of ricinus agglutinin with 2-mercaptoethanol in the presence of 0.5 M galactose the constituent peptide chains were separated by chromatography on a DE-52 column. The B′-chain passed through the column, whereas the A′-chain bound and was eluted with a salt gradient. The B′-chain was further purified by chromatography on a CM-52 column.The shortest chain, the A′-chain, was found to inhibit cell-free protein synthesis, whereas the B′-chain did not have this ability. On the other hand, the B′-chain was able to induce agglutination of erythrocytes when tested together with anti-ricinus agglutinin serum indicating that the B′-chains bind to the cells.Ouchterlony immunodiffusion tests with crude anti-ricin and anti-ricinus agglutinin sera revealed that the two constituent chains of ricinus agglutinin are immunologically partial identical and that they also show reaction of partial identity with both chains of the toxic lectin ricin.The data indicate that a similar structure-function relationship exists in ricinus agglutinin as in ricin. The reason for the much lower toxicity of ricinus agglutinin than of ricin in living animals is discussed.     

Cibacron Blue亲和层析及应用

以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ　Ｂｌｕｅ　Ｆ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法。实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ）,即蓖麻毒素Ａ链（ＲＴＡ）,苦瓜毒素（ｍｏｍｏｒｄｉｎ,ＭＴ）和Ｓａｐｏｒｉｎ,以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析显示三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光诸明显红移,提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合。将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕｅｄｅｘ。Ｂｌｕｅｄｅｘ亲和层析是一种经济有效,简单易行,便于在各类实验室中使用的蛋白质亲和层析技术。结果表明：在低盐溶液中ＲＴＡ和ＭＴ均可迅速地与Ｂｌｕｅｄｅｘ结合,而在高盐溶液中（０．６５ｍｏｌ／ＬＮａＣｌ）又极易被洗脱回收。这一技术用于免疫毒素的研究可有效地去除游离抗体,而不影响其杀伤活性。     

The effect of pH on the conformation and stability of the structure of plant toxin-ricin

The effect of pH on the conformation of ricin and its A- and B-chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A-subunit does not depend significantly on pH in the range pH 3-8, whereas ricin B-chain undergoes a structural transition at pH less than 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A-chain increases, whereas that of the B-chain decreases.     

Structure-toxicity Relationship in Subunit Structure of Ricin D

The re-formation of the single disulfide bond which linked two polypeptide chains of ricin D was studied. Ricin D was reoxidized preferentially by the air-oxidation of its reduced polypeptide chains in a yield of 74% with the recovery of full toxicity. Furthermore ricin D was completely regenerated from its compound with p-chloromercuribenzoate. It seems reasonable to assume that the toxicity of ricin D arises from the quaternary structure of ricin D molecule.     

Analysis of the proteins,glycoproteins and glycosaminoglycans of fibroblast adhesions to substratum

The focal adhesion preparations which remain attached to a glass substratum when fibroblast bodies are removed by a gentle stream of buffer have been analysed by gel electrophoresis coupled with other selective methods of analysis. The results are consistent with the presence of three classes of macromolecular components. (i) Muscle and associated proteins amongst which actin was abundant with significant amounts of tropomyosin, some myosin and traces of α-actinin. Some vimentin was present but no vinculin. We detected a major new protein component, as yet unidentified, with a molecular weight in the region of 50 000–55 000 which is not desmin or tubulin and could have an important function at the focal adhesion. (ii) Glycoproteins which are a specialised subset of those in the whole plasma membrane and included a family which bind ricin and therefore contain β-galactose end groups, together with a series having carbohydrate chains which bound neither ricin nor concanavalin A. The relative proportion of ricin-binding glycoproteins compared to concanavalin A-binding glycoproteins was higher than in whole plasma membranes. (iii) Glycosaminoglycans, with hyaluronate identified as the major component by column chromatography and its susceptibility to Streptomyces hyaluronidase.     

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.     

Iodination of Ricin D

Approximately five tyrosine residues of ricin D were iodinated preferentially under appropriate conditions probably forming diiodotyrosine. Iodination of this toxin carried out in 0.1 m phosphate buffer at pH 7.0 and 0°C for 60 min with a 20 fold molar excess of iodine per mole of protein, yielded a main component which appeared as a single band on polyacrylamide gel disc electrophoresis. Analysis of protein-bound radioactivity and the content of diiodotyrosine of ¹⁸¹I-labeled ricin D revealed that two tyrosine residues in the isoleucyl chain and three in the alanyl chain were substituted. The toxicity of iodinated ricin D decreased to one hundredth of that of native protein, However, the hemagglutinating activity of this protein was not affected by the iodination reaction.     

用琼脂糖亲和层析一步纯化蓖麻毒蛋白

本文报道了一种单用琼脂糖(Sepharose4B)来纯化蓖麻毒蛋白的快速简便的方法。我们发现在pH5的条件下,蓖麻毒蛋白和与其密切相关的蓖麻凝集素对琼脂糖的结合能力有很大的差别。在有0.2mol/LD-半乳糖存在下可将蓖麻毒蛋白从Sepharose上洗下,同样条件下蓖麻凝集素仍牢固地结合在柱上。从而经一步柱层析便可得到电泳纯的蓖麻毒蛋白。此法不需另行合成亲和胶,适合于蓖麻毒蛋白的大规模纯化。     

Conformation-dependent antigenic determinants in the toxic lectin ricin.

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.     

ONTOGENY OF CHICKEN HEMOGLOBIN II. CHROMATOGRAPHIC STUDY OF THE HETEROGENEITY OF HEMOGLOBIN IN DEVELOPMENT

Some properties of the carbonmonoxyhemoglobin (HbCO) from chicken embryos of ages 5, 10 and 15 days of incubation, from 1-day posthatching and from adult chickens have been investigated by chromatography on carboxymethylcellulose (CM-cellulose) column and by starch gel electrophoresis.
Chromatogram of the hemoglobin (Hb) from 5-day chicken embryos has shown that it consists of at least 6 components. Starch gel electrophoresis of each isolated component from the column in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown later that there are 3–4 embryonic type Hb components in 5-day embryos.
Chromatogram of the hemoglobin from adult chickens has shown that it consists of at least 4 components, but the examination of each isolated component from the column by electrophoresis in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown that there are 4–6 adult type Hb components in adults.
In ontogenic process, embryonic Hb type is detectable in embryos up to 15 days of incubation. Fetal Hb type, which is not detectable in adult chickens, can be first found in 10-day embryos.     

Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography

The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.