Functional binding analysis of human fibrinogen as an iron- and heme-binding protein

Human fibrinogen is a metal ion-binding protein, but its mechanism of binding with iron and heme has not been elucidated in detail. In this study, human fibrinogen was immobilized on CNBr-activated Sepharose 4B beads. The fibrinogen beads bound hemin (iron–protoporphyrin IX: PPIX) as well as iron ion released from ferrous ammonium sulfate (FAS) more efficiently than Sepharose 4B beads alone. Hemin bound to fibrinogen still exhibited pseudo-peroxidase activity. The affinity of fibrinogen binding to hemin, Sn–PPIX, Zn–PPIX and metal-free PPIX followed the order Sn–PPIX < metal-free PPIX < hemin < Zn–PPIX; PPIX bound more non-specifically to control beads. FAS significantly enhanced the binding of hemin to fibrinogen beads. These results suggest that human fibrinogen directly recognizes iron ion, the PPIX ring and metal ions complexed with the PPIX ring, and that the binding of hemin is augmented by iron ions.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Purification of TAXI-like Endoxylanase Inhibitors from Wheat (Triticum Aestivum L.) Whole Meal Reveals a Family of Iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

魔芋葡苷聚糖（KGM）凝胶为亲和层析载体与Sepharose4B的比较 …

将ＫＧＭ和Ｓｅｐｈａｒｏｓｅ ４Ｂ凝胶在相同条件下活化、偶联接上染料Ｃｉｂａｃｒｏｎ Ｂｌｕｅ Ｆ３ＧＡ制成了ＫＧＭ和Ｓｅｐｈａｒｏｓｅ ４Ｂ染料亲和吸附剂,并用来与牛血甭白蛋白（ＢＳＡ）作用,每毫升ＫＧＭ亲和吸附剂可吸附ＢＳＡ ２８ｍｇ,用ＮａＳＣＮ洗脱时间为８４．５％,而Ｓｅｐｈａｒｏｓｅ ４Ｂ染料样和吸附剂每毫升可吸附ＢＳＡ １５．３ｍｇ,用ＮａＳＣＮ洗脱时间收迷８１．７％,并对两种凝胶的染     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

固定化枯草杆菌色氨酰tRNA合成酶(英文)

为研究ｔＲＮＡＴｒｐ 与色氨酰ｔＲＮＡ合成酶（ＴｒｐＲＳ） 的相互识别及其结构、功能关系, 纯化了枯草杆菌ＴｒｐＲＳ并用溴化氰活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ 将ＴｒｐＲＳ固定化, 固定化ＴｒｐＲＳ的蛋白质回收率为９５ ．５ ％ , 活力回收率为３１．３％ 。研究了固定化ＴｒｐＲＳ的酶学性质, 其热稳定性和贮存稳定性方面均比液相ＴｒｐＲＳ有了较大的提高, 最适温度、最适ｐＨ 均有一定程度的增大, 工作稳定性良好。以固定化ＴｒｐＲＳ为亲和层析介质, 对含有２０ 个核苷酸随机序列、长度为５６ 个核苷酸的单链ＲＮＡ 随机库进行了３ 轮筛选,ＲＮＡ 群体亲和固定化ＴｒｐＲＳ的比例从４ ．３ ％ 上升至１４ ．７ ％ 。筛选得到了与ｔＲＮＡＴｒｐ 氨基酸接受茎类似的ＲＮＡ二级结构。实验结果表明固定化ＴｒｐＲＳ可以作为ＳＥＬＥＸ 亲和层析介质, 进行模拟ｔＲＮＡＴｒｐ 分子的ＲＮＡ 随机库的ＳＥＬＥＸ 筛选。     

Immobilization of L-glyceryl phosphorylcholine: isolation of phosphorylcholine-binding proteins from seminal plasma

The preparation of an affinity sorbent containing immobilized L-glyceryl phosphorylcholine for affinity chromatography of phosphorylcholine-binding proteins from seminal plasma is described. The ligand was coupled either after its maleinylation to poly(acrylamide-allyl amine) copolymer or directly to divinyl sulfone-activated Sepharose. The prepared phosphorylcholine derivative coupled to Sepharose was used for affinity chromatography of phosphorylcholine-binding proteins from bull and boar seminal plasma. Adsorbed proteins were specifically eluted with phosphorylcholine solution. Isolated phosphorylcholine-binding proteins were characterized by SDS electrophoresis and HPLC with reversed phase. Composition of the boar phosphorylcholine-binding fraction obtained by affinity chromatography on immobilized L-glyceryl phosphorylcholine was compared with that eluted from immobilized heparin by the phosphorylcholine solution. No phosphorylcholine-binding proteins were found in human seminal plasma.     

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.     

枯草杆菌TrpRS的固定化及亲和RNA筛选

为研究 t RNATrp与色氨酰 - t RNA合成酶 ( Trp RS)的相互识别及其结构与功能的关系 ,纯化了枯草杆菌 Trp RS,并用溴化氰活化的 Sepharose4B将 Trp RS固定化 ,固定化 Trp RS的蛋白回收率为 95.5% ,活力回收率为 31 .3% .研究了固定化 Trp RS的酶学性质 ,其热稳定性和贮存稳定性方面均比液相 Trp RS有了较大的提高 ,最适温度、最适 p H均有一定程度的增大 ,工作稳定性良好 .以固定化 Trp RS为亲和层析介质 ,对含有 2 0个核苷酸随机序列 ,长度为 56个核苷酸的单链RNA随机库进行了三轮筛选 .实验结果表明 ,固定化 Trp RS可以作为 SELEX亲和层析介质 ,进行模拟 t RNATrp分子的 RNA随机库的 SELEX筛选 .     

Anhydroelastase: enhanced affinity toward product-type ligands revealed by affinity chromatography

Anhydroelastase was effectively isolated by a single operation of affinity chromatography from a complex mixture produced by phenylmethylsulfonylation and alkaline treatment of porcine pancreatic elastase. The adsorbent used for the chromatography was 6-aminohexanoyl-trialanine, which corresponds to a product of elastase action, immobilized on Sepharose 4B. Successful resolution by the operation indicated that this immobilized ligand possesses the highest affinity for anhydroelastase among various proteins including regenerated elastase in the mixture. Comparative affinity chromatography on immobilized anhydroelastase and on immobilized native elastase further confirmed the stronger interaction of anhydroelastase with the product-type peptides. Immobilized anhydroelastase was also found to be useful in the purification and search for naturally occurring proteinase inhibitors.     

Isolation and characterization of root nodule proteins from lupin

A group of root nodule-specific plant proteins (nodulins) has been isolated from yellow lupin (Lupinus luteus) by immunoaffinity chromatography. The cytoplasmic nodule protein extract was initially enriched in nodulins on a column with immobilized IgG fraction. It was then purified by chromatography on Sepharose 4B - bound IgG against uninfected root proteins and finally on Sepharose 4B - bound IgG against Rhizobium lupini proteins. Rocket immunoelectrophoresis showed that the nodulin preparation did not react with antibodies against root or bacterial proteins. SDS gel electrophoresis of lupin nodulins revealed at least 23 polypeptides ranging in Mr, from 7,000 to 70,000, probably representing protein subunits.     

Isolation and characterization of two major serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid P component

Two major serum components from the dogfish, Mustelus canis, have been isolated using affinity chromatography. Both proteins bind to an AH-Sepharose 4B-phosphorylcholine affinity matrix in the presence of Ca2+ and are eluted from the column by EDTA. Upon readdition of Ca2+ to the eluted proteins, the two proteins can be separated by passage through a column of Sepharose CL-4B. The first protein, C-reactive protein, passes through the Sepharose CL-4B column in the presence of Ca2+ whereas the second protein, serum amyloid P component, remains bound. The serum amyloid P component is then eluted from the Sepharose CL-4B in pure form by EDTA. The dogfish C-reactive protein isolated by the phosphorylcholine affinity matrix precipitates with pneumococcal C-polysaccharide and with a synthetic derivative of bovine serum albumin to which phosphorylcholine is covalently attached. The precipitation is inhibited by either EDTA or by phosphorylcholine. Dogfish C-reactive protein has a molecular weight of approximately 250,000 with dimeric subunits of Mr = 50,000. Upon addition of beta-mercaptoethanol these dimeric subunits dissociate to two identical monomeric subunits of Mr = 25,000. The protein cross-reacts immunologically with goat antisera prepared against rabbit C-reactive protein. The dogfish serum amyloid P component has a molecular weight of at least 250,000 with monomeric subunits of Mr = 25,000. Cross-reactivity of the amyloid P component with the C-reactive protein could not be shown. However, NH2-terminal sequence analysis of the first 20 amino acids showed some homology. The relationship of dogfish C-reactive protein to the C-reactive proteins in Limulus polyphemus and in rabbits and humans is discussed.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

Hexadecanedionic acid-sepharose 4B: A new tool for preparation of albumin-depleted plasma

Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.     

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column.

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.     

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.