A homogeneous enzyme immunoassay with avidin-ligand conjugate as the enzyme-modulator

A homogeneous enzyme immunoassay (EIA) based on the immunomodulation of an avidin-ligand conjugate and the inhibition of pyruvate carboxylase is described. The conjugation of the ligand, 5,5-diphenylhydantoin (DPH), to avidin does not affect avidin's capacity to bind biotin or inhibit pyruvate carboxylase. The DPH-avidin conjugate and free DPH were shown to compete for a limited number of antibody sites. The interaction of anti-DPH with the DPH-avidin conjugate sterically inhibited enzyme inactivation. Enzyme activity was correlated with DPH concentrations in the therapeutic range found in serum.     

Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.     

Author index for volume 202

A procedure is described for a simple two-step purification of human liver propionyl-CoA carboxylase. The method is based on acid and carbon tetrachloride extraction to remove other biotin carboxylases followed by an 800-fold purification through biotin-pretreated, monomeric avidin-Sepharose 4B-CL with elution of active enzyme using a biotin gradient. The enzyme had a sedimentation coefficient of 17.4 S and polyacrylamide gel electrophoresis after reduction and alkylation revealed two nonidentical polypeptide chains of 75,000 and 60,000 M_r. The heavier chain was identified as the biotin-containing subunit by electrophoresis after avidin binding.     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.     

Biochemical and Biological Characterization of a New Oxidized Avidin with Enhanced Tissue Binding Properties

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff''s base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected ¹¹¹In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.     

Hydrolytic enzymes conjugated to quantum dots mostly retain whole catalytic activity

BackgroundTagging a luminescent quantum dot (QD) with a biological like enzyme (Enz) creates value-added entities like quantum dot–enzyme bioconjugates (QDEnzBio) that find utility as sensors to detect glucose or beacons to track enzymes in vivo. For such applications, it is imperative that the enzyme remains catalytically active while the quantum dot is luminescent in the bioconjugate. A critical feature that dictates this is the quantum dot–enzyme linkage chemistry. Previously such linkages have put constraints on polypeptide chain dynamics or hindered substrate diffusion to active site, seriously undermining enzyme catalytic activity. In this work we address this issue using avidin–biotin linkage chemistry together with a flexible spacer to conjugate enzyme to quantum dot.MethodsThe catalytic activity of three biotinylated hydrolytic enzymes, namely, hen egg white lysozyme (HEWL), alkaline phosphatase (ALP) and acetylcholinesterase (AChE) was investigated post-conjugation to streptavidin linked quantum dot for multiple substrate concentrations and varying degrees of biotinylation.ResultsWe demonstrate that all enzymes retain full catalytic activity in the quantum dot–enzyme bioconjugates in comparison to biotinylated enzyme alone. However, unlike alkaline phosphatase and acetylcholinesterase, the catalytic activity of hen egg white lysozyme was observed to be increasingly susceptible to ionic strength of medium with rising level of biotinylation. This susceptibility was attributed to arise from depletion of positive charge from lysine amino groups after biotinylation.ConclusionsWe reasoned that avidin–biotin linkage in the presence of a flexible seven atom spacer between biotin and enzyme poses no constraints to enzyme structure/dynamics enabling retention of full enzyme activity.General significanceOverall our results demonstrate for the first time that streptavidin–biotin chemistry can yield quantum dot enzyme bioconjugates that retain full catalytic activity as native enzyme.     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Label-free amplified bioaffinity detection using terahertz wave technology

A new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidin molecules. Amplified detection of avidin-biotin binding is obtained on supported membranes composed of biotin layers on quartz surface, which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to the octadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin and biotin-avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was able to detect less than 10.3 ng/cm2 avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry and reflectometry techniques. Further improvement is underway using highly refractive beads together with appropriate surface chemistry. This newly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand-analyte affinity binding.     

A chip-based method for studying the effects of exogenous proteins on neuronal axons

It has been reported that the activity of protein improved when it was adsorbed inside the pores of mesoporous silica (MPS). The current study investigated the activity of immobilized avidin to the biotin on MPS with various pore sizes (diameter=2.4-45.0 nm). The binding amount of immobilized avidin to biotin is 123 to 160 ng biotin/10 μg avidin on 2.7- to 5.4-nm pore MPS, but that on 12- to 45-nm pore MPS was markedly decreased (33-42 ng biotin/10 μg). Moreover, the binding amount was approximately 2- and 3-fold higher on the glycidoxypropyl (Gly)-functionalized 5.4- and 45-nm pore MPS in comparison with methyl (Me)-functionalized 5.4- and 45-nm pore MPS, respectively. Furthermore, avidin immobilized in native and Gly-grafted 45-nm pore MPS retained more than 70% and 50% binding activity to biotin, respectively, after incubating at 90°C for 3 h. In contrast, the activity was greatly reduced in the native and Gly-grafted 5.4-nm pore MPS under the same conditions (<36.9%). The immobilization also protected against effects of 0.01 M HCl and 50% MeOH; all of immobilized avidin proteins showed high activity (>50%) with biotin compared with that observed with free avidin (MeOH [<18.2%] and HCl [<32.7%]).     

The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T₄) was prepared by N-acylation of N-(3-aminopropyl)biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T₄ conjugate with one or simultaneously with two binding proteins with affinity to Bt or T₄ in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T₄ was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T₄. The Bt-T₄ conjugate also formed a specific equimolar complex with T₄-binding human globulin (TBG) by the same mechanism as that for T₄. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T₄ conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T₄. The intact T₄ competitively inhibited this interaction after its addition to the system. Bt-T₄ also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu³⁺-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T₄ immobilized on a solid phase. Free T₄ inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T₄ complex was actively bound to the T₄-antibody, but had practically no interaction with serum T₄-binding proteins, including TBG. Probably, nonspecific interactions of the T₄ residue with streptavidin in its complex with Bt-T₄, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T₄-binding proteins of blood serum. The Bt-T₄ stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.     

Insecticidal activity of recombinant avidin produced in yeast

An expression construct encoding chicken (Gallus gallus) avidin was assembled from amplified fragments of genomic DNA. Recombinant, functional avidin was produced in Pichia pastoris, with yields of up to 80 mg/l of culture supernatant. The recombinant avidin had similar insecticidal activity to egg white avidin when assayed against larvae of a lepidopteran crop pest, cabbage moth (Mamestra brassicae), causing >90% reduction in growth and 100% mortality when fed in optimised diets at levels of 1.5 μM and 15 μM (100 ppm and 1000 ppm wet weight of recombinant protein). The recombinant protein was also highly toxic to a hemipteran pest, the pea aphid (Acyrthosiphon pisum), when fed in liquid artificial diet, causing 100% mortality after 4 days when present at concentrations ≥3.8 μM (0.25 mg/ml, 250 ppm). Mortality was dose-dependent, with an estimated LC₅₀ of 2.1 μM. Toxicity to A. pisum was prevented by biotin supplementation of diet. In contrast, avidin had no significant effects on the survival of cereal aphid (Sitobion avenae) at concentrations up to 30 μM in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to recombinant avidin in the diet through proteolysis of the ingested protein, which would allow recovery of bound biotin.     

Evidence for the biosynthesis of selenobiotin

Chemically synthesized selenobiotin is, like sulfur biotin, able to bind to avidin. This observation was used to help identify biologically synthesized selenobiotin as an excretion product of $\text{Phycomyces blakesleeanus}$. The identification of [⁷⁵Se]selenobiotin was based on the highly specific binding of biotin to avidin used as an affinity ligand to Sepharose, on its release from the complex by proteolytic treatment, and its chromatographic behavior relative to [¹⁴C]biotin standards. These results represent the first evidence of a biological synthesis of a heterocyclic ring that contains selenium in place of sulfur.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.     

Malonyl-CoA Decarboxylase from Streptomyces erythreus: Purification,properties, and possible role in the production of erythromycin

Malonyl-CoA decarboxylase was purified (800-fold) from an erythromycin-producing strain of Streptomyces erythreus using DEAE-cellulose, Sephadex G-100, SP-Sephadex, and gel filtration with Sephadex G-75. The molecular weight of the native enzyme was 93,000 as determined by gel filtration and the subunit molecular weight was 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, suggesting an α₂ subunit composition for the native enzyme. Evidence is presented that during the purification procedure and storage a proteolytic cleavage occurred resulting in the formation of 30- and 15-kDa peptides. The enzyme showed a pH optimum of about 5.0 whereas the vertebrate enzyme showed an optimum at alkaline pH. The enzyme decarboxylated malonyl-CoA with a K_m of 143 μm and V of 250 nmol min^?1 mg^?1. For the decarboxylation of methylmalonyl-CoA this enzyme showed the opposite stereospecificity to that shown by vertebrate enzyme; the (R) isomer was decarboxylated at 3% of the rate observed with malonyl-CoA while the (S) isomer was not a substrate. Neither avidin nor biotin affected the rate of malonyl-CoA decarboxylation, suggesting that biotin is not involved in catalysis. Acetyl-CoA and free CoA were found to be competitive inhibitors. Propionyl-CoA, butyryl-CoA, succinyl-CoA, and methylmalonyl-CoA showed little inhibition, and neither thiol-directed reagents nor chelating agents inhibited the enzyme. High ionic strength and sulfate ions caused reversible inhibition of the enzymatic activity. Under two different cultural conditions the time course of appearance of malonyl-CoA decarboxylase was determined by measuring the enzyme activity and the level of the enzyme protein by an immunological method using rabbit antibodies prepared against the enzyme. In both cases the increase and decrease in the decarboxylase correlated with the rate of production of erythromycin, suggesting a possible role for this enzyme in the antibiotic production.     

A modular design of low‐background bioassays based on a high‐affinity molecular pair barstar:barnase

High‐affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K_D) of the molecular pair, with avidin:biotin being the state‐of‐the‐art molecular pair (K_D ～ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high‐affinity protein pair, barstar:barnase (K_D ～ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non‐negligible background due to the non‐specific binding. A quantitative assessment of the non?specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin‐based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non‐specific binding, showing good prospects for high‐sensitivity rare biomolecular event nanoproteomic assays.     

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).     

Biomphalaria glabrata: lysozyme activities in the hemolymph, digestive gland, and headfoot of the intermediate host of Schistosoma mansoni.

Lysozyme (mucopeptide N-acetylmuramylhydrolase EC 3.2.1.17) activity has been found in the hemolymph, digestive gland, and headfoot extracts of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. Partial purification of the bacteriolytic enzyme was attained by gel chromatography on Sephacryl S-200 and active lytic fractions were concentrated by Amicon filtration. The properties of the lytic enzymes from the three tissue extracts were identical. Enzyme activity was determined by the rate of lysis of cell wall suspension of Micrococcus lysodeikticus. Lysis of the cell walls was accompanied by a release of reducing sugar groups and N-acetylhexosamines. The enzyme was stable to heating at 100 C for 2 min and had an optimum activity at pH 4.5 to 5.0 in 0.066 M glycylglycine buffer. Low concentrations (5 mM) of NaCl, KCl, and LiCl increased the activity of the enzyme, whereas high concentrations (25 mM) of the same ions caused about 50% inhibition of the enzyme activity. MgCl₂ and CaCl₂ also inhibited the enzyme activity. Addition of 1 mM EDTA or EGTA resulted in about a twofold increase in enzyme activity. Double reciprocal plots of enzyme velocities and substrate concentrations yielded an apparent Michaelis-Menten constant (K_m) of 0.05 ± 0.01 mg/ml of M. lysodeikticus.     

The biotin requirement of HeLa cells

We have examined the effect of alterations in the biotin content of the medium on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of the HeLa cells. The inclusion in the growth medium of avidin, which almost irreversibly binds with biotin (K_d, 10^?15 M), results in an increase in cellular biotin content and biotin enzyme activity over that seen when the cells are grown in a biotin-depleted medium. The addition of avidin-bound biotin to the growth medium led to a forty-fold increase in cellular biotin when compared to the inclusion of an equivalent amount of free biotin in the medium. HeLa cells are able to internalize avidin-bound biotin. Biotin is released from this complex to function as the prosthetic group of biotin enzymes. HeLa cells do have a nutritional requirement for biotin.