Synthesis of tag introducible (3-trifluoromethyl)phenyldiazirine based photoreactive phenylalanine

An efficient synthesis of tag introducible (3-trifluoromethyl)phenyldiazirine based phenylalanine derivatives is described. Alkylation of a chiral glycine equivalent with a spacer containing (3-trifluoromethyl)phenyldiazirinyl bromides enables us to create photoreactive L-phenylalanine derivatives. After introduction of biotin at the spacer, the biotinylated and photoreactive amino acid was applied for L-amino acid oxidase and incorporated into a substrate binding site. These compounds will be powerful tools not only for photoaffinity labeling to elucidate properties of bioactive peptides but also as trifunctional photophors to introduce a ligand skeleton.     

Investigation of spacer length effect on immobilized Escherichia coli pili-antibody molecular recognition by AFM

The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Poly(ethylene glycol) (PEG) and Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to silicon wafer surfaces for the purpose of improving the orientation of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a variety of linear polymers were used to covalently attach the antibodies to silicon surfaces. Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical composition at each reaction step. The effect of spacer length in improving the specificity of immobilized antibody was investigated by attaching E. coli on the end of an AFM tip. The distribution of unbinding force and rupture distance from the force-distance curves obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the incidence of interactions by up to 90%. J600 proved to be the most effective spacer overcoming the steric hindrance seen with direct immobilization of antibody. In addition, the force spectroscopy reveals the elementary force quantum of E. coli-antibody to be 0.3 nN.     

Probing the adenosine receptor with adenosine and xanthine biotin conjugates

Biotin-containing analogs of a potent agonist (N6-phenyladenosine) and a potent antagonist (1,3-dipropyl-8-phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para-position of the phenyl ring. Two biotin conjugates of N6-phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter-chain derivative was more potent in inhibiting binding of N6-[3H]cyclohexyladenosine to rat cerebral cortical membranes (Ki of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3-dipropyl-8-phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.     

Biotinyl-estradiol derivatives in enzyme immunoassays: structural requirements for optimal antibody binding

The use of the avidin/biotin complex in immunoassays is well documented. No comprehensive studies, however, are available on the structural requirements of the linkage between biotin and small molecules to get an optimal antigen-antibody interaction. We have synthesized seven different biotinylated estradiol derivatives. They were evaluated in an antibody- and in an antigen-immobilized enzyme immunoassay system. All three derivatives lacking a spacer group were useless for use in immunoassays, demonstrating the importance of a long distance between the biotin- and estradiol-moiety. In addition, the chemical structure of the linkage at the site of attachment to the steroid skeleton is very important for the antibody recognition: it may either be rigid but identical to that one used in the immunogen (6-carboxymethyloxime), or must be structurally flexible as exemplified by a 6-amido-linkage. A rigid structure (hydrazone) different from that of the immunogen absolutely prevents antibody binding.     

Impact of spacers on the hybridization efficiency of mixed self-assembled DNA/alkanethiol films

The immobilization of DNA strands is an essential step in the development of any DNA biosensor. Self-assembled mixed DNA/alkanethiol films are often used for coupling DNA probes covalently to the sensor surface. Although this strategy is well accepted, the effect of introducing a spacer molecule to increase the distance between the specific DNA sequence and the surface has rarely been assessed. The major goal of this work was to evaluate a number of such spacers and to assess their impact on for example the sensitivity and the reproducibility. Besides the commonly used mercaptohexyl (C(6)) spacer, a longer mercapto-undecyl (C(11)) spacer was selected. The combination of both spacers with tri(ethylene)glycol (TEG) and hexa(ethylene)glycol (HEG) was studied as well. The effect of the different spacers on the immobilization degree as well as on the consecutive hybridization was studied using surface plasmon resonance (SPR). When using the longer C(11) spacer the mixed DNA/alkanethiol films were found to be more densely packed. Further hybridization studies have indicated that C(11) modified probes improve the sensitivity, the corresponding detection limit as well as the reproducibility. In addition two different immobilization pathways, i.e. flow vs. diffusion controlled, were compared with respect to the hybridization efficiency. These data suggest that a flow-assisted approach is beneficial for DNA immobilization and hybridization events. In conclusion, this work demonstrates the considerable impact of spacers on the biosensor performance but also shows the importance of a flow-assisted immobilization approach.     

A streptavidin mutant useful for directed immobilization on solid surfaces.

A streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of streptavidin on solid surfaces. This streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of streptavidin-based solid-phase assay systems.     

Site-directed immobilization of antibody onto solid surfaces for the construction of immunochip

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity,i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidincoated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.     

Structure-function relationships in immobilized chymotrypsin catalysis

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.     

Structure-function relationships in immobilized chymotrypsin catalysis

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.     

Spin labeling and kinetic studies of a membrane-immobilized proteolytic enzyme.

Membrane-based bioreactors can greatly influence the rate and extent of chemical reactions and consequently lower the costs associated with the corresponding engineering processes. However, in order to progress in this area, greater understanding of the relationship of the structure and function of bioreactor systems is required. In this study, a proteolytic enzyme, papain (EC 3.4.22.2), was covalently coupled onto the surface of a vinyl alcohol/vinyl butyral copolymer (PVB) membrane employing either glutaraldehyde (GA) or 1,1'-carbonyldiimidazole (CDI). Various kinetic and performance properties of the immobilized papain were studied. It was found that these characteristics of the membrane-bound papain depended on the immobilization method. The CDI-immobilized papain bioreactor was used, although the apparent Michaelis constant, Km, of the CDI-immobilized papain was larger than that of the GA-immobilized enzyme. In separate experiments, a six-carbon spacer was also used between the membrane support and the covalently-linked enzyme. It was found that the insertion of the spacer reduced the disturbance of the enzyme system, resulting in a decreased Km, which was now closer to the value for the free enzyme. Electron paramagnetic resonance (EPR) techniques of spin labeling were used for the first time to examine the conformational change and the active site structure of an enzyme covalently immobilized to a membrane. The structural changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme.     

Influence of azide incorporation on binding affinity by small papain inhibitors

In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by ‘click’ chemistry, in this particular case by copper-free, strain-promoted azide–alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.     

Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma

Studies in our laboratory and others indicate that biotin is released from biotinylated proteins in vivo and in vitro in human plasma. Using immunoglobulin G (IgG) as the model protein and four different biotinylating reagents, we investigated the mechanism of release. All of the biotin bonds shared an amide link to the carboxyl group of biotin but differed in the chemical links (amide, thioether, and hydrazone) between spacer arm and the various functional groups on IgG. Biotinylated IgG was incubated with phosphate-buffered saline, plasma, or plasma treated to either inactivate enzymes or remove all macromolecules. Released biotin was separated from bound biotin by ultrafiltration and quantitated by avidin-binding assay. As judged by high-performance liquid chromatography, greater than 95% of the released avidin-binding activity was biotin. We infer that the amide bond between the biotin and the spacer arm rather than the bond attaching the spacer to the protein was cleaved. Sodium dodecyl sulfate gel electrophoresis detected no proteolytic degradation of biotinylated IgG. Neither heat inactivation of plasma nor ultrafiltration of plasma to remove macromolecules completely eliminated biotin cleavage. We conclude that cleavage of biotin from protein occurs by both enzymatic and nonenzymatic mechanisms.     

Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.     

Biotin derivatives of D-Phe-Pro-Arg-CH2Cl for active-site-specific labeling of thrombin and other serine proteinases

Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation of three biotin derivatives of the thrombin-specific inhibitor D-Phe-Pro-Arg-CH2Cl containing increasing lengths of the spacer connecting biotin. Reaction of the peptide with biotin-succinimidyl esters and purification by conventional chromatography yielded the compounds in 91-96% purity. The biotin-labeled inhibitors bound avidin with stoichiometries of 0.88-1.02 mol biotin compound/mol avidin subunits and irreversibly inactivated human thrombin with stoichiometries of 0.89-1.10 mol inhibitor/mol thrombin. Comparison of the three inhibitors by Western blotting indicated that a > or = 7- to 14-atom spacer was needed for sensitive (approximately 10 ng) detection of thrombin, with the derivative lacking a spacer only weakly detected because of its greatly reduced affinity for avidin. Application of the compounds to identify catalytically active products of factor Xa-catalyzed human prethrombin 1 activation in the absence of the protein cofactor, factor Va, allowed the direct observation of transient, low levels of the active intermediate, meizothrombin des-fragment 1, in addition to thrombin. Formation of this intermediate is concluded to reflect an intrinsic property of factor Xa activation of prethrombin 1 that is modulated by factor Va. The methods developed for preparation and characterization of the biotin-labeled inhibitors may be applicable to other tripeptide chloromethyl ketones, and the reagents can be employed for labeling of serine proteinases of diverse substrate specificity.     

Molecular cloning of a putative agglutination/immobilization antigen located on the surface of a novel agglutination/immobilization serotype of Cryptocaryon irritans

A surface agglutination/immobilization antigen was purified from the novel agglutination/immobilization serotype (serotype G37) of the ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes. Serum from fish immunized with C. irritans theronts had agglutination/immobilization activity against theronts in vitro. However, fish and rabbit antisera raised against serotype G32 (reported previously) caused little agglutination/immobilization of serotype G37 theronts. Immunological analysis indicated that the 37 kDa theront surface membrane protein may be the agglutination/immobilization antigen of this serotype. The full-length 37 kDa antigen cDNA contained 1171 base pairs, encoding a 331-amino acid protein with hydrophobic N- and C-termini, which are characteristically found in proteins containing a C-terminal glycosylphosphatidylinositol anchor. In addition, the genetically characterized nucleotide sequences of the first internal transcribed spacer region of ribosomal DNA of these 2 serotypes were compared. The internal transcribed spacer rDNA sequence of serotype G32 was identical to that of isolates from Pingtung, Taiwan, and from the USA. On the other hand, the sequences of serotype G37 were not identical to those of any C. irritans isolate.     

A protected biotin containing deoxycytidine building block for solid phase synthesis of biotinylated oligonucleotides.

The synthesis of a modified 2'-deoxycytidine-3'-O-phosphoramidite carrying an N-t-butylbenzoyl protected biotin on a long polar spacer arm attached to the 4-N position is described. The presence of the bulky lipophilic t-butylbenzoyl protecting group enables the direct solid phase synthesis of biotinylated oligoribonucleotides and a variety of analogues in high yield without modification of the biotin moiety. Biotinylated antisense oligonucleotides incorporating this new derivative allow convenient isolation and purification of ribonucleic acid-protein complexes. The kinetics of biotin binding to streptavidin agarose is facilitated by the long polar spacer arm.     

Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules.     

Mechanistic investigation of smart polymer-protein conjugates

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

Peptide Biotinylation with Amine-Reactive Esters: Differential Side Chain Reactivity

Miller, B. T., T. J. Collins, M. E. Rogers and A. Kurosky. Peptide biotinylation with amine-reactive esters: differential side chain reactivity. Peptides 18(10) 1585–1595, 1997.—N-hydroxysuccinimide (NHS) esters of biotin are reported to react specifically with amino groups of peptides and proteins. However, we have found that these reagents can readily acylate other functional groups in specific peptide sequences under relatively mild conditions. We have extended our inquiry of sequence-dependent acylation by evaluating the reactivity of a variety of commonly employed biotinylation reagents typically used for amino group modification. These included the p-nitrophenyl ester of biotin, NHS-esters of biotin containing aminohexanoic acid spacer arms, and a sulfonated NHS-biotin ester that contained a disulfide bond within its spacer. The decapeptide [D-Lys⁶]gonadotropin releasing hormone was employed as a model peptide. Reaction products were characterized by high-performance liquid chromatography, amino acid compositional analysis, reaction with hydroxylamine, and mass spectrometry. In addition to the O-acylation of Ser⁴ and Tyr⁵ in this peptide, we have also identified a novel biotinylation of the Arg⁸ side chain.     

UV resonance Raman study of streptavidin binding of biotin and 2-iminobiotin: comparison with avidin

UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin-biotin complex and new data from the avidin-2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43.