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.     

Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization

A new biotin-carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000-000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, and binding was accompanied by strong quenching. In the present study the specific quenching of 'biotin-4-fluorescein' was utilized to measure (strept)avidin concentrations (0.2-2 nM) by the extent of fluorescence quenching at 8 nM ligand concentration. Adsorption of (strept)avidin to the assay tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Virtually the same specific response to avidin and streptavidin was also observed with commercial 'fluorescein-biotin', except that >10 h incubation times were required. The slow association of 'fluorescein-biotin' was attributed to the anti-cooperative binding which is due to the much longer spacer as compared to 'biotin-4-fluorescein'. The third ligand tested in this study was 'biotin-4-FITC' which was analogous to 'biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate residue. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avidin-bound state. In conclusion, the new ligand 'biotin-4-fluorescein' appeared to be the most general and convenient probe: quenching was most pronounced and linearly dependent on (strept)avidin concentrations, the dose response for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation time.     

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.     

The highly dynamic oligomeric structure of bradavidin II is unique among avidin proteins

Bradavidin II is a biotin‐binding protein from Bradyrhizobium japonicum that resembles chicken avidin and bacterial streptavidin. A biophysical characterization was carried out using dynamic light scattering, native mass spectrometry, differential scanning calorimetry, and isothermal titration calorimetry combined with structural characterization using X‐ray crystallography. These observations revealed that bradavidin II differs from canonical homotetrameric avidin protein family members in its quaternary structure. In contrast with the other avidins, bradavidin II appears to have a dynamic (transient) oligomeric state in solution. It is monomeric at low protein concentrations but forms higher oligomeric assemblies at higher concentrations. The crystal structure of bradavidin II revealed an important role for Phe42 in shielding the bound ligand from surrounding water molecules, thus functionally replacing the L7,8 loop essential for tight ligand binding in avidin and streptavidin. This bradavidin II characterization opens new avenues for oligomerization‐independent biotin‐binding protein development.     

A monovalent streptavidin with a single femtomolar biotin binding site

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.     

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.     

A fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid

Avidin and biotin can be sensitively and accurately quantitated using the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS). In the presence of avidin, the fluorescence of 2,6-ANS is blue shifted with a large increase in quantum yield. Biotin binding causes complete displacement of the bound fluorophore with concomitant quenching of the fluorescence. The fluorometric monitoring of the displacement of 2,6-ANS can be used as a facile method of measuring the biotin-avidin interaction. 2,6-ANS displacement gives the same stoichiometry as the method using 4'-hydroxyazobenzene-2-carboxylic acid. Our initial studies of an affinity-purified avidin revealed that, of the four binding sites on the avidin tetramer, a mean of three remain available for biotin (or dye) binding; this finding highlights a caveat concerning the use of affinity-purified oligomeric-binding proteins with multiple sites. As compared with previous fluorescence methods, the use of 2,6-ANS gives high sensitivity without the necessity of preparing and purifying a covalent avidin conjugate. In addition, the present method;:is potentially more sensitive than those based on optical absorbance; uses a probe that has increased stability and a larger Stokes shift compared with fluorescein; is not subject to protein interference; and gives accurate results over a wide range of 2,6-ANS and avidin concentrations.     

Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.     

A small-molecule-linked DNA–graphene oxide-based fluorescence-sensing system for detection of biotin

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin–DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3′ to the 5′ terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5–20 nmol/L. The detection limit for biotin is 0.44 nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein–small molecule ligand binding.     

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole‐labeled biotin

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole‐labeled biotin. BODIPY‐FL‐aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position‐specifically incorporated into Trp120 position of streptavidin by four‐base codon method. On the other hand, carbazole‐labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole‐labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole‐labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole‐labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.     

Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin

The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T(m) = 107.4 +/- 0.3 degrees C) than avidin (T(m) = 83.5 +/- 0.1 degrees C) or bacterial streptavidin (T(m) = 75.5 degrees C). AVR4/5 also exhibits a high affinity for biotin (K(d) approximately 3.6 x 10(-14) m) comparable to that of avidin and streptavidin (K(d) approximately 10(-15) m). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed.     

Interaction between biotin lipids and streptavidin in monolayers: formation of oriented two-dimensional protein domains induced by surface recognition

Highly specific ligand-receptor interactions generally characterize surface recognition reactions. Such processes can be simulated by streptavidin-biotin-specific binding. Biotin lipids have thus been synthesized, and their interaction with streptavidin (or avidin) at the air-water interface was directly shown by measurement of surface pressure isotherms and fluorescence microscopy. These proteins interact with the biotin lipid monolayer via specific binding or nonspecific adsorption. Both phenomena were clearly distinguished by use of the inactivated form of streptavidin. The binding of fluorescein-labeled streptavidin to monolayers was also directly observed by fluorescence microscopy. The fluorescence of the protein domains is directly related to the state of polarization of the exciting light. This anisotropy can only be explained by the formation of oriented two-dimensional biotin lipid-streptavidin domains.     

Zebavidin - An Avidin-Like Protein from Zebrafish

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.     

Bifunctional avidin with covalently modifiable ligand binding site

The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.     

Streptavidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence

While the binding of biotin by streptavidin does not appear to be cooperative in the traditional sense of altered binding strength, it has been suggested that it may be cooperative in terms of differential structural changes in the protein. In this work we present intrinsic tryptophan fluorescence data as evidence of a cooperative structural change. The technique involves examination of the differences in fluorescence emission corresponding to distinct tryptophan populations accompanying protein-ligand binding. Specifically we note that the 335 nm emission population (i.e. more hydrophobic) saturates prior to the saturation of the 350 nm emission population commonly used in the standard binding activity assay. We also note that the wavelength of maximum emission, total integrated fluorescence emission and full width at half maximum during the titration of ligand into streptavidin also reach saturation before the expected 4:1 stoichiometric end point. This suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand.     

Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.

Covalent DNA-streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA-streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded DNA fragment or an enzyme and (ii) low-molecular weight biotin derivatives ("modulators") were coupled stepwise with the hybrid molecules. Modulators were D-biotin, aminobiotin, and biotin-fluorescein conjugate as well as a lysine-rich 10mer peptide, containing a biotin and a fluorescein substituent. These modulators were chosen to affected the hybridization properties of the DNA-streptavidin conjugates. As investigated by surface-plasmon resonance and microplate solid-phase hybridization measurements, D-biotin, biotin-fluorescein, and aminobiotin decreased the efficiency of hybridization with complementary, surface-bound oligonucleotides to a varying extent. The basic peptide increased the conjugate's hybridization efficiency. Moreover, it was demonstrated in two examples how modulators can be utilized as additional functional domains of streptavidin-based conjugates. First, fluorescein-containing modulators were used as hapten groups, allowing a sensitive detection by means of specific antibodies directed against the modulator. Second, the biotinylated peptide was used as a carrier molecule to attach multiple fluorogenic lanthanide-chelate groups to the streptavidin conjugate, enabling its sensitive detection by time-resolved fluorometry. The applicability of this kind of bioconjugation strategy to generate sensor-probes for gene detection assays was demonstrated.     

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.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Easily reversible desthiobiotin binding to streptavidin,avidin, and other biotin-binding proteins: uses for protein labeling,detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.     

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.