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.     

Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.     

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.     

Biotinylphallotoxins: preparation and use as actin probes

We describe the synthesis of four phalloidin derivatives conjugated with biotin. An aminomethyldithiolane derivative of ketophalloidin was used as a reactive starter compound, and biotin residues were coupled to this molecule either directly, separated by spacer chains comprised of one or two glycyl residues, or of a 12-atom long chain constructed from succinic acid and hexamethylendiamine. Although all products still displayed a high affinity for F-actin, as seen in competition experiments with [3H]-demethylphalloidin, only the one with the longest spacer (BHPP) showed specific and high-affinity decoration of actin filaments in permeabilized cells, in conjunction with FITC-coupled avidin and fluorescence microscopy. Combined with gold-streptavidin, BHPP decorated the actin filament system at the light and electron microscopic level faithfully and with satisfactory density. Actin filaments polymerized in vitro from purified protein were not as densely labeled as had been expected. However, in all these experiments the new phalloidin probe, when combined with avidin or streptavidin, yielded clear and highly specific labeling of F-actin. Therefore, this system is useful to identify and localize actin unambiguously in microfilaments, independent of actin antibodies, and should facilitate double-label experiments on cytoskeletal components at the ultrastructural level.     

Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli K1

The N-propionylated group B meningococcal polysaccharide mimics a unique bactericidal epitope on the surface of group B meningococci and Escherichia coli K1. This was confirmed when both the above organisms were able to absorb the bactericidal antibodies from a mouse-anti-N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate serum. By using affinity columns it was possible to divide the conjugate antiserum into three distinct populations of both group B polysaccharide cross-reactive and non-cross-reactive antibodies, one of which contained most of the bactericidal activity. The cross-reactive (IgG1) antibodies were absorbed by an affinity column in which the group B polysaccharide was linked to the solid support by a long spacer arm, thereby isolating a population of non-cross-reactive (IgG1) antibodies. Surprisingly the above column also retained another population of non-cross-reactive (IgG2a) and (IgG2b) antibodies which contained most of the bactericidal activity. These latter antibodies were not absorbed by a similar group B polysaccharide-affinity column in which a short spacer arm was employed. Thus the above experiments not only effected a separation of highly bactericidal antibodies but also provided evidence that the long spacer arm is functional in the binding of the bactericidal antibodies to the affinity column. This indicates that the bactericidal epitope is mimicked by the group B polysaccharide in the presence of the long spacer arm, which supports the hypothesis that the epitope is polysaccharide-associated and is probably intermolecular in nature.     

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.     

Synthesis Of Oligodeoxyribonucleotides Containing an Aliphatic Amino Linker Arm at Selected Adenine Bases and Derivatization with Biotin

Abstract

The chemical synthesis and characterization of oligodeoxyribonucleotides with a “linker arm” attached to the 8-position of some adenine bases are presented. Derivatizatlon with biotin is described and evaluation of these modified DNA fragments as nonradioactive hybridization probes is briefly discussed.     

Polymer-bound reagents for the introduction of spacer-modified biotin labels

We have developed a method for the chemoselective introduction of spacer modified biotin labels into unprotected multi-functional amines. A range of novel biotin spacer conjugates attached to a polymer-bound sulfonamide anchor was prepared using established amide bond forming procedures. After chemical transformation of the attachment site by alkylation, the resulting reactive species were utilized as N-selective polymer-supported biotinylation reagents. The labeled compounds, obtained in good to excellent yield and purity, are free of residual biotin and possess a custom tailored distance from the immobilization site being especially suited for the immobilization on streptavidin-functionalized dextran layers of surface plasmon resonance detector chips. In addition, derivatives displaying a phenyl group were synthesized in order to demonstrate the versatility of the procedure for the simultaneous introduction of spacer-modified biotin and a UV-light absorbing moiety. The formation of biotin sulfoxides in the presence of in situ generated peroxides was investigated and is discussed. Our results suggest that this derivatization technique is a useful addition to the existing biotin labeling protocols.     

Introduction of aliphatic amino and hydroxy groups to keto steroids using O-substituted hydroxylamines.

(Aminooxy)butyl- and -hexylamines and alcohols were synthesized by the Ing-Manske modification of the Gabriel synthesis. The aminooxy group of these heterobifunctional spacer reagents is a far more powerful nucleophile than the amino or hydroxy group because of the oxygen atom adjacent to the amino group (alpha-effect). The aminooxy group reacts readily with keto groups while the amino or hydroxy (or other) group remains free for further reactions. These excellent heterobifunctional spacer reagents were used here to derivatize keto steroids in an alkaline alcoholic solution. Using the described, general, and easy one-step synthesis, aliphatic amino or hydroxy groups with a spacer arm have been introduced to testosterone, 6-ketoestradiol, and cortisol.     

Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum

A new time-resolved fluoroimmunoassay (TR-FIA) of testosterone in serum is described, using a biotinylated testosterone tracer, with a long spacer arm between biotin and testosterone, coupled to the C3 of the testosterone: a biotinylaminodecane carboxymethyloxime testosterone. This tracer affords a great sensitivity of the standard curve, because a amount of 0.3 pg of testosterone can be significantly measured on the testosterone standard curve. The "functional" sensitivity is at least equal to 21 pg/ml of serum. The specificity of the assay is insured by a celite chromatographic step on new minicolumns before immunoassay. The variation coefficient of inter-series reproducibility measured on low and normal testosterone levels in untreated and testosterone treated hypogonadal men were between 2.17 and 5.07%. The accuracy test, (overload and dilution tests) gave satisfying results. Moreover, in a comparison with GCMS, it appeared that the correlation coefficient was 0.992 and no significant difference could be exhibited between the two methods. Consequently, this specific, sensitive reproducible and easy to use method is well suited to the measurement of testosterone in clinical and pharmacological conditions.     

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.     

Synthesis of biotin-containing phosphoramidite linker with polyether spacer arm

A phosphoramidite linker unit, based on glycerol backbone and containing a biotin residue attached through a tetraethylene glycol spacer arm, was synthesized. DMTr-Glycidol and tetraethylene glycol were used as starting materials. After conversion of one of hydroxy groups in tetraethylene glycol into an amino group, the epoxy cycle in DMTr-glycidol was opened by this amino alcohol, resulting in the corresponding ether and some quantity of secondary amine. After attaching of biotin residue to the ether followed by phosphitylation, the desirable linker was obtained. The structure of the linker was confirmed by (1)H-(1)H COSY, (1)H-(13)C HSQC, (1)H-(13)C HMBC, (1)H-(15)N HSQC, and (1)H-(15)N HMBC spectra. The resulted phosphoramidite linker unit is suitable for use in common DNA synthesizers. This approach can be used for preparation of various modifiers containing reporter groups attached to the primary amino function using conventional procedures.     

Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures

We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.     

Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes.

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.     

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.     

Ligands for insulin receptor isolation

Biotinylated insulins are bivalent molecules having the ability to bind to insulin receptors on the one hand and to "avidins" on the other. In order to be useful as ligands for insulin receptor isolation, biotinylated insulins must be developed that have the capacity to bind simultaneously to both and insulin receptor. The present investigation addresses this problem. A series of biotinylated and dethiobiotinylated insulins has been prepared in which the distance between the biotin carboxyl group and the insulin varies from 7 to 20 atoms. These compounds form complexes with succinoylavidin. The dissociation rates (K-1) of these complexes have been determined from the [14C]biotin exchange assay. The dissociation kinetics of most of these complexes are biphasic, and the kinetic constants reported are those corresponding to the slow rate. Ligands containing dethiobiotin dissociate more rapidly than the corresponding biotin derivatives. The interposition of a spacer arm substantially decreases the rate of dissociation. The [14C]biotin exchange assay could not be used with streptavidin complexes of the above ligand since biotin dissociates more rapidly from streptavidin than from succinoylavidin. However, the relative dissociation rates of a series of ligands could be determined and were as follows: 6-(dethiobiotinylamido)-hexanoic acid greater than dethiobiotinyl-A1-insulin greater than biotinylinsulin greater than biotinyl-A1-insulin greater than biotinyl-A2-insulin. Dethiobiotin and its amide failed to form complexes with streptavidin. The affinity of the ligands for insulin receptors was determined by measuring their ability to stimulate 14CO2 formation from [1-14C]glucose in rat epididymal adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The hydrophobic properties of beta-glycoprotein from the blood serum of pregnant rats

During immunoelectrophoresis in the presence of tween-80, triton X-100 and ammonium sulfate blood serum beta-glycoprotein of pregnant rats migrated along with beta-globulins as a main single band; its minor components in zones of alpha- and gamma-globulins were not detected. beta-glycoprotein was completely absorbed by phenyl sepharose in the absence of ligand as well as when the spacer arm for phenyl group was short. When the phenyl group was linked with the template through a long spacer arm, three froms of beta-glycoprotein with different immunoelectrophoretic mobility were detected after absorbtion with phenyl sepharose. Hence, beta-glycoprotein is hydrophobic and is represented by alpha-, beta- and gamma-forms in blood plasma of pregnant rats.