N-hydroxysulfosuccinimide Ester Research Articles

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors.

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca++ ] is consistent with parasite killing specifically via action on one or more Ca++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca++ transport and may be starting points for new antimalarial drugs.

Spectrophotometric method for the quantitative assay of N-hydroxysulfosuccinimide esters including extinction coefficients and reaction kinetics

A quantitative spectrophotometric method has been developed for the analysis of N-hydroxysulfosuccinimide (sulfo-NHS), a chromophore with a maximum absorbance at 268 nm. The extinction coefficients were determined between pH 6.0 and 8.0 and found to vary in a nonlinear manner. This spectrophotometric profile is not present in its esters which however release an equimolar amount of sulfo-NHS when they react with nucleophilic groups or hydrolyze in aqueous solution. This fact facilitates the determination in solution of the concentration and purity of bis(sulfosuccinimidyl) suberate (BS3) used as a model, as well as the examination of hydrolysis and aminolysis half-lives in different reaction conditions, these parameters being valuable in optimization of the use of the active esters.

Detection of Small Molecules Using Long-Range Surface Plasmon Polariton Waveguides

An optical biosensor utilizing long-range surface plasmon-polariton waveguides is discussed. This paper first reviews the technology and its various applications such as protein sensing, cell detection, and disease diagnostics, and then presents results on the detection of small molecules using the biosensor. The surface was functionalized with 16-mercaptohexadecanoic acid and activated using carbodiimide chemistry. Covalent attachment of N-hydroxysulfosuccinimide ester (Sulfo-NHS) of molecular weight ∼200 Da produced a significant response with a signal-to-noise ratio of ∼81 as a result of molecular monolayer formation. Since carbodiimide chemistry is a protein coupling approach commonly used to functionalize biosensors, a calibration scheme for extracting the actual amount of attached protein based on biosensor responses to NHS is proposed.

Bioconjugated Fluorescent Zeolite L Nanocrystals as Labels in Protein Microarrays

Zeolite L nanocrystals, as inorganic host material containing hydrophobic fluorophore N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide in the unidirectional channels, are developed as new labels for biosensor systems. The external surface of the particles is modified with carboxylic acid groups for conjugation to primary amines of biomolecules such as antibodies. Anti-digoxigenin (anti-DIG) is selected to be immobilized on zeolite L via N-hydroxysulfosuccinimide ester linker. Together with DIG, it serves as a good universal binding pair for diverse analyte detection owing to the high binding affinity and low background noise. The conjugates are characterized by the dynamic light scattering technique for their hydrodynamic diameters and by enzyme-linked immunosorbent assay for antigen-antibody binding behavior. The characterizations prove that anti-DIG antibodies are successfully immobilized on zeolite L with their binding activities maintained. The microarray fluorescent sandwich immunoassay based on such nanocrystalline labels shows high sensitivity in a thyroid-stimulating hormone assay with the lower detection limit down to the femtomolar range. These new fluorescent labels possess great potential for in vitro diagnostics applications.

Polysialylation Increases Lateral Diffusion of Neural Cell Adhesion Molecule in the Cell Membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.

Comprehensive Study of the Formation and Reaction of a Tethered N-Hydroxysulfosuccinimidyl Ester Used to Covalently Tether Proteins to Surfaces

A fundamental understanding of surface-reaction kinetics and mechanisms is essential to making progress on rational design of complex surface architectures used in applications as varied as biosensor devices and organic electronic devices. We present recent progress on the understanding of the carbodiimide coupling reaction frequently used to form a surface-tethered active N-hydroxysulfosuccinimide ester and the subsequent substitution of the active ester with various nucleophiles in solution. These studies were carried out using surface-enhanced Raman spectroscopy (SERS) and show that the carbodiimide coupling reaction is first order in carbodiimide concentration and zero order in N-hydroxysulfosuccinimide concentration. This reaction proceeds with a second-order rate constant of 0.062 mM-1s-1 at about 20 °C. The tethered active ester was then reacted with histidine or glucose oxidase. The reaction with histidine was found to be first order in the histidine solution concentration, indicating a simple SN2...

Extracellular lysines on the plasmodial surface anion channel involved in Na+ exclusion.

The human malaria parasite, Plasmodium falciparum, induces an unusual ion channel, the plasmodial surface anion channel (PSAC), on its host red blood cell (RBC) membrane. PSAC has a broad selectivity with permeability to anions, sugars, amino acids, purines, and certain vitamins, suggesting a role in nutrient acquisition by the intracellular parasite. Permeating solutes cover a range of molecular sizes and may be either neutral or carry a net negative or positive charge. Despite this broad selectivity, PSAC must efficiently exclude Na+ to avoid osmotic lysis of infected RBCs in the bloodstream. Here, we used amine-reactive N-hydroxysulfosuccinimide esters to probe PSAC's unusual selectivity. PSAC permeation rates, measured with both a kinetic osmotic lysis assay and single-channel patch-clamp, irreversibly decrease after treatment with these reagents. Sequential labelings with different esters and the effects of their chain length suggest that PSAC has multiple lysine residues near its extracellular pore mouth and that inhibition occurs via steric hindrance of its pore by the amide-linked side chain. When combined with the effects of pH on permeation, these findings implicate a combination of cation repulsion by pore mouth charges and a weak binding site for permeant solutes in PSAC's broad selectivity yet effective exclusion of Na+.

Studies of Surface Coverage and Orientation of DNA Molecules Immobilized onto Preformed Alkanethiol Self-Assembled Monolayers

DNA molecules are attached onto carboxylate-terminated alkanethiol self-assembled monolayers (SAMs) preformed at gold surfaces via the N-hydroxysulfosuccinimide (NHS)/1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) cross-linking reaction. Cyclic voltammetry, quartz crystal microbalance (QCM), and atomic force microscopy (AFM) were used to probe the surface coverage and molecular orientation of the immobilized DNA molecules. The DNA attachment is attributed to the formation of amide bond between the carboxylate groups and the amino groups on the DNA bases since the possibility of nonspecific adsorption of DNA onto preformed SAMs or NHS ester monolayers was studied and excluded. Our voltammetric results indicate a significant blockage of the sites originally available in the alkanethiol SAMs for facile heterogeneous electron transfer by the attached DNA molecules. QCM provided a semiquantitative measurement of the amount of immobilized DNA. The AFM images, for the first time, revealed th...

Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells.

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans-Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.

Preparation of oligonucleotide-biotin conjugates with cleavable linkers.

A procedure is presented for preparing an oligonucleotide-biotin conjugate that is chemically cleavable through the reduction of a disulfide bond within the linker. Conjugation involves reaction of a primary amine with an N-hydroxysulfosuccinimide ester linked to biotin. The oligonucleotide can be liberated from streptavidin agarose containing immobilized conjugate under mild conditions (neutral pH, 50 mM dithiothreitol). This cleavable conjugate is useful for affinity purification applications.

Biotinylation: a nonradioactive method for the identification of cell surface antigens in immunoprecipitates.

A nonradioactive method was employed to detect different cell membrane antigens on human polymorphonuclear granulocytes, monocytes and platelets. We compared the reactivity of one monoclonal antibody, N1III10, assumed to be Fc gamma RII-specific by functional assays, with other well-characterized monoclonal antibodies and human sera. Intact cells were incubated with biotin N-hydroxysulfosuccinimide ester which preferentially reacts with lysine residues in polypeptides. Biotin-labeled cells were lysed and the antigen was isolated from the cell lysate by immunoprecipitation with the antibody bound to Protein A-Sepharose. The precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and visualized by a streptavidin-alkaline phosphatase system with a suitable substrate. Using this biotin-labeling system we could show that N1III10 detects a 40 kDa antigen on monocytes and platelets, comparable to that expected of Fc gamma RII monoclonal antibodies.

Multiple folate transport systems in L1210 cells

Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the μ m transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the μ m transporter and the n m transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the μ m transporter (43 kDa) and the n m transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the n m transporter with peptide: N-glycosidase F produced a smaller component (32 kDa); the μ m transporter, conversely, was unchanged by this procedure. When the μ m transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the n m transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 μ m transporter is a non-glycosylated, integral membrane protein, while its n m counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.

A nondestructive method for peptide bond conjugation of antigenic haptens to a diphtheria toxoid carrier, exemplified by two antisera specific to acetolactate synthase

A method for the preparation of an activated protein carrier is described: Protein carboxyl groups are transformed into N-hydroxysulfosuccinimide esters, a structure that will react with primary amino groups under amide bond formation. Although the activated ester is unstable under aqueous conditions, a significant amount of hapten molecules can be bound covalently to the carrier under very mild conditions. Ligands can be peptides or other molecules possessing a primary amino group. The method avoids the risk of ligand polymerization and no derivatization of the ligand prior to conjugation is needed. Residual unreacted ligand molecules can therefore be recovered in their native form by size exclusion chromatography. The method was used to conjugate two synthetic sugar beet acetolactate synthetase (E.C. 4.1.3.18) peptides to diphtheria toxoid. Antibodies were raised against both of the conjugates. The specificity of these antibodies against sugar beet acetolactate synthetase was verified using immunoblotting, ELISA, and immunoprecipitation.

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)—II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl- N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited ≈ 10–100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nefand the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10μg) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: > 16,000, l: > 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nefcarriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.

Development of technology for linking photosensitizers to a model monoclonal antibody

A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3–4 sulfhydryl residues per carrier molecule, and introduction of sulfo- m-maleimidobenzoyl- N-hydroxysulfosuccinimide ester residues to the monoclonal (3–4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided.

Biotinylation: a simple method for labelling complement component C8 with preservation of functional activity

Biotinylation of human C8 with the water-soluble biotin derivative biotinylamidohexanoic acid, N-hydroxysulfosuccinimide ester is an excellent method for labelling this terminal complement component with preservation of its functional activity. The biotinylated product can be detected both in native form and also following its incorporation into the terminal complement complexes. Detection assays include Western blotting, crossed immunoblotting, ELISA, and immunocytochemistry. Biotinylation is an attractive alternative method for labelling C8 and may be used for detecting and quantifying C8 and C5b-9 complexes in their soluble and membrane-bound forms.

Biotinylation: An alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates

A method has been developed in which the conventional radioiodine label is replaced by non-radioactive biotin in studies involving the immunoprecipitation and analysis of cell surface antigens. The labelling reagent, d-biotinyl- N-hydroxysulfosuccinimide ester (NHSS-biotin), reacts preferentially with lysine residues in polypeptides and possibly also with free amino-groups on carbohydrates and lipids. The reagent can be used as a cell surface label, does not interfere with antigen-antibody interactions and allows labelled molecules to be detected with high sensitivity using streptavidin-peroxidase conjugates. The target antigens of a range of monoclonal antibodies to human cell surface components have been identified using this procedure.

Reactions of N-hydroxysulfosuccinimide active esters.

N-Hydroxysulfosuccinimide esters are reactive functional groups employed in a variety of protein modification reagents, especially cross-linking reagents. For these compounds, hydrolysis is the most important reaction competing for reaction of the esters with nucleophilic groups in proteins. We have employed model compounds to investigate the rates of hydrolysis of N-hydroxysulfosuccinimide esters and their reactions with the alpha-amino group and the side chains of naturally occurring amino acids, under conditions comparable to those used for protein modification studies. The rats of hydrolysis observed were found to be very low, as compared with their rates of reaction with nitrogen nucleophiles found in proteins. Further, within the ranges investigated, the rate of aminolysis was observed to increase more rapidly than the rate of hydrolysis with increasing pH or with increasing temperature. Four amino acid side chains and the alpha-amino group were found to react measurably with N-hydroxysulfosuccinimide esters. At pH 7.4 and room temperature, the order of reactivity was found to be N alpha-Cbz-histidine greater than N alpha-Cbz-lysine approximately phenylalanine (alpha-amino group) much greater than N-acetylcysteine approximately N-acetyltyrosine; however, the acylimidazole adduct formed with the side chain of histidine was found to be a transient product, subject to hydrolysis or reaction with another nucleophile.