Covalent immobilization of the estrogen receptor to a cationic N-hydroxysuccinimide ester derivative of agarose

Covalent immobilization of the soluble estrogen receptor from a rabbit uterus to N-hydroxysuccinimide ester derivative of agarose is shown. At first, the condition for the immobilization reaction was examined. The non-immobilized receptor was extracted with 0.4 M NaCl-containing medium. Sixty seven to 80% of the input receptor were immobilized within 30 min at 0 degrees C in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4). The immobilized [3H]estradiol(3,17 beta-dihydroxy-1,3,5(10)-estratriene)-receptor complex was stable for at least 24 h. The optimum pH for immobilization was 7.4. Ca2+ or Na+ ions in the reaction media decreased the yields in immobilization of the receptor to the reagent with an electrostatically positive spacer arm. Next, influences of immobilization on the receptor were examined. The dissociation rate of [3H]estradiol from the immobilized receptor was a little slower than that from the native receptor. The estrogen-free immobilized receptor was saturated by incubating with 10 nM [3H]estradiol for 10 h at 0 degrees C in 0.1 M HEPES (pH 7.4). From Scatchard plot analysis, it was found that the hormone binding affinity in the immobilized receptor decreased to approximately one-fourth of that in the native receptor.     

Covalent immobilization of the estrogen receptor to an electrostatically neutral N-hydroxysuccinimide ester derivative of agarose

Immobilization of the estrogen receptor to the N-hydroxysuccinimide ester of succinylethylenediaminocarboxymethyl agarose (Reagent B) is described and compared with that to the charged N-hydroxysuccinimide ester derivative (Reagent A), previously described. The time course for immobilization was examined. Thirty-six percent of the input receptor was immobilized within 1 h. The optimum pH in immobilization is 7.0-7.4. The dissociation rate of [3H]estradiol(3,17 beta-1,3,5(10)-estratriene) from the [3H]estradiol-receptor complex immobilized to Reagent B was similar to that in Reagent A. The receptor immobilized to Reagent B was saturated with estradiol at 5 h. The [3H]estradiol concentration necessary for saturation was 10 nM. The dissociation constant (KD) for the receptor immobilized to Reagent B was 0.95 X 10(-9) M.     

Characterization of the cooperative cross-linking of doxorubicin N-hydroxysuccinimide ester derivatives to water soluble proteins.

Protein-anthracycline interactions have been examined by using reactive N-hydroxysuccinimide ester derivatives of doxorubicin. These compounds cross-link to lysine epsilon-amino groups with high efficiency and offer the possibility for structural studies of protein-anthracycline complex formation by using gel filtration, ultracentrifugation and spectrophotometric methods. The results are in accordance with association of anthracycline to the hydrophobic ligand binding cavities of serum albumin. The results for proteins not having hydrophobic domains (IgG, serum transferrin, lactotransferrin, ovotransferrin) suggest that complex formation is cooperative and involves two steps: initial self-association of anthracycline into aggregated structures and subsequent binding of protein at the aggregate surface. With serum transferrin, anthracycline self-association makes possible the assembly of stable nanometer-sized protein-anthracycline particles held together by non-covalent bonds. This reaction, which is highly reproducible and efficient, may have applications in the field of development of anthracycline carrier systems.     

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.     

Covalent protein immobilization on glass surfaces: application to alkaline phosphatase

Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558-1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4+/-0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent.     

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.     

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.     

Solid-state conjugation of proteins with hydrophobic compounds in non-denaturing conditions. I. Acylation of proteins by dansyl proline using a polymeric N-hydroxysuccinimide ester

A method of conjugating protein molecules under native conditions with water-insoluble hydrophobic compounds is developed. It permits very water-insoluble acids to be gently coupled to the primary amines on proteins or other biopolymers. For this purpose we synthesized a polymer (co-polymer of N-hydroxymaleimide and N-vinylpyrrolidone cross-linked by benzidine) which swells equally well in water and in organic solvents. Hydrophobic substances are first activated by esterification to this polymer in organic solvent and then conjugated to protein by acylation in aqueous medium at pH 8.0-8.5. Thus, the contact of native protein with organic co-solvent may be completely avoided. The application of this approach is demonstrated by labeling trypsinogen and plasminogen with dansyl proline.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.     

Covalent immobilization of proteins and peptides for solid-phase sequencing using prepacked capillary columns

The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described. Capillary tubes with an internal volume of about 30 microliters are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs. Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodi-imide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass. Optimization of attachment conditions is described. The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded. This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (less than 50-1000 pmol) or macrosequencing (1-50 nmol).     

Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy

A genetically modified form of the human DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.     

Laccase mediator system for activation of agarose gel: Application for immobilization of proteins

Cross-linked Sepharose beads were treated with laccase–TEMPO system for oxidation of the primary alcohol groups on the sugar moieties. Optimal activation conditions using Trametes versicolor laccase were at pH 5 and 22 °C, giving an aldehyde content of 55 μmol g⁻¹ Sepharose with 28 units g⁻¹ of laccase and 12.5 mM TEMPO. The activated Sepharose was used for immobilization of trypsin as model protein. Highest degree of immobilization was obtained at pH 10.5 but the activity yield was only 31% of that loaded on the gel. The yield of gel bound trypsin activity was increased to 76% (corresponding to about 43 U g⁻¹ Sepharose) when the immobilization was performed in the presence of trypsin inhibitor, benzamidine. The immobilization yields were comparable to that obtained on the matrix activated using sodium periodate (containing 72 μmol aldehyde per g Sepharose). Recycling and storage of the immobilized trypsin preparations showed high stability of the enzyme bound to laccase–TEMPO activated gel.