Article pubs.acs.org/biochemistry
Self-Assembly of a Functional Triple Protein: Hemoglobin-AvidinHemoglobin via Biotin−Avidin Interactions Serena Singh and Ronald Kluger* Davenport Chemical Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada M5S 3H6 S Supporting Information *
ABSTRACT: Hypertension resulting from vasoconstriction in clinical trials of cross-linked tetrameric (α2β2) human hemoglobins implicates the extravasation of the hemoglobins into endothelia where they scavenge nitric oxide (NO), which is the signal for relaxation of the surrounding smooth muscle. Thus, we sought an efficient route to create a larger species that avoids extravasation while maintaining the oxygenation function of hemoglobin. Selectively formed cysteine-linked biotin conjugates of hemoglobin undergo self-assembly with avidin into a stable triple protein, hemoglobin-avidin-hemoglobin (HbAvHb), which binds and releases oxygen with moderate affinity and cooperativity. The triple protein is likely to be stabilized by interactions of each constituent hemoglobin (pI 6.9) with the oppositely charged avidin (pI 10.5) as well as the strong association of the biotin moieties on hemoglobin with avidin. first developed an efficient route for specific biotinylation of Hb utilizing a biotin-maleimide reaction with the two accessible thiols of each β-cys-93. The resulting Hb-biotin conjugates associate with avidin to produce the desired assembly of proteins. While these are stabilized by the specific interactions of avidin and biotin, electrostatic interactions should further stabilize the assembly based on the contrasting isoelectric points of the two proteins: pI(Hb) 6.9 and pI(avidin) 10.5. This interpretation is consistent with the charge-directed association in the assembly of avidin and the globular protein ovalbumin.10
T
he potential clinical utility of cell-free native hemoglobin (Hb) as a circulating oxygen carrier cannot be realized because it is readily excreted and in the course of circulation can produce renal damage from iron deposition as well as hypertension.1,2 Chemical modification strategies were developed that render Hb stable for circulation as a tetramer, avoiding dissociation into its constituent dimers.3 While stabilized tetramers are large enough to avoid renal filtration, clinical trials revealed that they produce hypertension, most likely as a result of their removal of endothelial nitric oxide upon extravasation. An increase in the functional size of the stabilized tetramer by the coupling of tetramers to one another is likely to prevent extravasation and consequent scavenging of nitric oxide.4 While Hb-based oxygen carriers (HBOCs) from glutaraldehyde-based cross-linking5 are in principle large enough to prevent extravasation, the method of preparation necessarily produces partially modified materials that remain subject to extravasation. To reduce or eliminate heterogeneity resulting from nonspecific modification, highly selective chemical protein modification processes have been developed. The resulting materials that appear to be successful in avoiding vasoconstriction include Hb bis-tetramers6 and Hb-albumin clusters.7 The preparations of those materials are limited in scalability due to operationally complex steps and multiple purification processes. We reasoned that an alternative approach based on protein− ligand affinity could be more efficient for protein−protein coupling. In particular, we reasoned that such a coupling could result from adaptation of the high affinity of biotin and avidin. Also, while avidin antibodies exist in circulation, addition of avidin does not produce a clinical effect.8 Avidin has four biotin-binding sites,9 making it a potential scaffold for efficient protein−protein coupling where the proteins are initially coupled to biotin-related conjugates at specific sites. Thus, we © 2016 American Chemical Society
■
EXPERIMENTAL SECTION Protein Sources. Purified human hemoglobin A was obtained from Oxygenix Co. Ltd. Avidin from egg white was obtained from BioShop. Biotin-maleimide (N-biotinoyl-N′-(6maleimidohexanoyl)hydrazide) was purchased from SigmaAldrich. Fumaryl cross-linked Hb (α99-fumaryl-α99, β2) was prepared according to the procedure by Walder et al.11 Trimesoyl cross-linked Hb (α2, β82-trimesoyl-β82) was prepared by published methods from tris(3,5-dibromo-salicyl)trimesate.12 Self-Assembly of Hb and Avidin. Absorbance at 700 nm was acquired for Hb species/avidin and Hb/lysozyme mixtures (5 μM each protein in 1 mL of the specified buffer) through the use of a Cintra 40 UV−vis spectrometer. The Hb species were in the CO-bound state. Independent studies with added inositol hexaphosphate (IHP) (2 equiv, 2.58 μL of a 4 mM solution in H2O) were conducted in the same manner. In a separate experiment, a 1/1 mixture of native Hb and avidin was Received: March 7, 2016 Revised: April 26, 2016 Published: April 29, 2016 2875
DOI: 10.1021/acs.biochem.6b00215 Biochemistry 2016, 55, 2875−2882
Article
Biochemistry
Figure 1. Absorbance at 700 nm, associated with solution turbidity, resulting from formation of 1/1 mixtures of native or cross-linked Hb and avidin in buffers of varying pH and ionic strength. Buffers: phosphate, pH 7.4 (I = 24 mM); Tris, pH 8.3 (I = 3 mM); tris, pH 9.0 (I = 1 mM); borate, pH 9.2 (I = 54 mM).
fixated using 13 equiv of glutaraldehyde (11 μL of a freshly prepared 0.05 M solution in water) for every 1.0 equiv of Hb (0.2 mM in 210 μL of 10 mM HEPES buffer, pH 8.0). The reaction was terminated by the addition of 100 μL of 1 M Tris (pH 8.0), and then the mixture was concentrated through a 30 kDa cutoff filter. Biotinylation of Hb. To native Hb (0.075 μmol, 0.075 mM in 1 mL of 50 mM phosphate, pH 6.5) in the carbon monoxidebound state was added the biotin-maleimide cross-linker (20 equiv, 24 μL of a 62.5 mM stock solution in DMSO). The Hbcontaining solution was stirred at room temperature for 1 h in a crimp-sealed vial flushed with CO(g). Excess reagent was removed from the protein solution by four cycles of centrifugation (14000g, 5 min) through a filter (30 kDa cutoff) followed by dilution to the original volume with 50 mM phosphate (pH 6.5). The biotinylated Hb species were analyzed by reverse-phase HPLC (see the Supporting Information) through the use of a 330 Å C-4 Vydac column (4.6 mm × 250 mm) with a solvent gradient from 20% to 60% acetonitrile/water with 0.1% trifluoroacetic acid. The eluent was monitored at 220 nm. Observed drifts in retention times may be attributed to solvent evaporation over time (solvents were mixed off-line) and variations in column equilibration time, temperature, and degassing. The composition of the protein associated with the peaks was investigated by isolation of the fractions and analysis using electrospray ionization (ESI) high-resolution mass spectrometry (AIMS Lab, Department of Chemistry, University of Toronto; see the Supporting Information). Biotinylated fumaryl/trimesoyl cross-linked Hb were prepared by the same method. Thermal Stability of Biotinylated (Non-Cross-Linked) Hb. The UV−vis spectrometer Peltier-controlled cell was maintained at 60.0 °C, and protein solutions in the carbon monoxide-bound state (10 μM in 1 mL of 0.01 M phosphate buffer, pH 6.5) were cooked for 10 min. The absorbance spectra (500 to 700 nm) were acquired at 1 min intervals. Hb-Avidin Conjugation. To a solution of avidin (0.019 μmol, 86 μL of a 0.22 mM solution in a 50 mM phosphate buffer, pH 6.5) was added the solution of modified Hb (3 equiv, 0.057 μmol, 710 μL of a 0.08 mM solution in 50 mM phosphate, pH 6.5). The resulting solution was stirred at room temperature for 1 h in a crimp-sealed vial flushed with carbon monoxide. The final products were stored under an atmosphere
of carbon monoxide at 4 °C. The resulting conjugates were analyzed by size-exclusion HPLC using a Superdex G-200 HR size-exclusion column (10 mm × 300 mm) and a Tris-HCl (37.5 mM, pH 7.4) elution buffer containing magnesium chloride (0.5 M). The eluent was monitored at 280 nm. Conjugates of fumaryl/trimesoyl cross-linked Hb and avidin were prepared by the same method. Conjugates with 1/1 Hb/ avidin were made by the combination of 1 equiv of biotinylated Hb to approximately 5 equiv of avidin. The final products contained less than 1% percent methemoglobin. Occupancy Assay of Non-Cross-Linked Hb-Avidin Conjugate with HABA Dye. To independent solutions of native avidin and the Hb-avidin conjugate (4 and 1 μM avidin, respectively, in 1.0 mL of a 50 mM phosphate buffer, pH 6.5) was added 4′-hydroxyazobenzene-2-carboxylic acid (HABA) (1−20 μL of a 5 mM solution). The reference cell contained either avidin or the hemoglobin-avidin conjugate at the same concentration. The absorption spectra from 200 to 700 nm were acquired using a UV−vis spectrometer. An increase in absorbance at 500 nm (ε = 34.5 M−1) is associated with bound HABA.13 Binding curves were derived from the changes in absorbance. The same assay was performed on nonconjugated biotinylated Hb as a control. Native PAGE of Hb-Avidin Conjugates. Two-dimensional Tris-HCl polyacrylamide gels contained 6% or 12% separating gel (pH 8.8) and 4% stacking gel (pH 8.8). The buffered solution containing the sample was adjusted to pH 6.8, and running buffer was adjusted to pH 8.3. Gels run with reverse polarity are noted specifically. Finished gels were stained with Coomassie Brilliant Blue. A comprehensive procedure for native polyacrylamide gel electrophoresis (PAGE) was followed.14 Oxygen-Binding Properties of Non-Cross-Linked HbAvidin Conjugate. The material prepared using an excess of avidin (1/1 Hb/avidin conjugate) was utilized for oxygenation studies. The oxygen pressure at half-saturation (P50) and Hill’s coefficient of cooperativity at half-saturation (n50) were determined using a Hemox Analyzer15 with the sample maintained at 27 °C. Hb samples (5 mL, 7 μM) prepared in phosphate buffer (0.01 M, pH 7.4) were oxygenated prior to analysis by being stirred under a stream of oxygen with photoirradiation for 1.5 h at 0 °C. Samples were then transferred to a cell connected to the Hemox Analyzer for 2876
DOI: 10.1021/acs.biochem.6b00215 Biochemistry 2016, 55, 2875−2882
Article
Biochemistry
Figure 2. Absorbance at 700 nm, associated with solution turbidity, of 1/1 mixtures of fumaryl cross-linked Hb and avidin in buffers of varying pH and ionic strength.
native Hb/avidin mixtures, native Hb/lysozyme mixtures failed to produce an effect. The absorbance changes recorded with cross-linked Hb/lysozyme mixtures were minimal, with the absorbance at 700 nm reaching a maximum of only 0.044 OD when the lysozyme/Hb ratio was 7/1 (Figure 5). The unique effect in the observed cross-linked Hb/avidin synergy suggests the occurrence of charge compensation, as has been previously discussed.10 These structures may be organized microspheres.10 Thus, questions remain about their properties in circulation and potential applications.16,17 We tested if acylation drives aggregation by combining native Hb and avidin nonspecifically using glutaraldehyde. Native Hb treated with glutaraldehyde produces soluble polymeric species.18 In contrast, polymerization of these oppositely charged proteins produces turbidity in the sample. Upon concentration, this produces a mass that shatters into crystalline-like shards upon agitation (Figure 6). Therefore, we developed an alternative based on site-specific modification. Self-Assembly of Hb-Biotin-Avidin-Biotin-Hb. We observe that a similar association between biotinylated Hb and avidin is effective in the formation of bis-tetramers of Hb associated with avidin as a specific assembly, namely, Hb-biotinavidin-biotin-Hb (designated as HbAvHb). Biotinylation of Hb. Native carbon monoxide-bound Hb (and subsequently fumaryl/trimesoyl cross-linked Hb) was treated with 20 equiv of the biotin-maleimide reagent (Scheme 1). Conducting protein modification under an atmosphere of carbon monoxide minimizes the probability of methemoglobin formation. Two biotin molecules were incorporated per Hb tetramer, as determined by mass analysis (see the Supporting Information), with the β-subunit cys-93 residues as the expected sites of modification. Proteins were manipulated in the stable carbon monoxide-bound state to minimize the probability of methemoglobin formation. Thermal Stability of Biotinylated (Non-Cross-Linked) Hb. Modification of the solvent-accessible thiols of cysteine residues of the β-subunits affects the heat stability of the tetramer. The carbon monoxide-bound protein provides a welldefined substrate to evaluate the perturbation. Subjecting the biotinylated tetramer to higher temperatures reveals that the modification decreases the protein’s intrinsic heat stability. Native Hb maintains its structural stability upon being heated at 60 °C for 10 min (Figure 7, top panel). In contrast, biotinylated Hb denatures under the same conditions, and a turbid solution
acquisition of the oxygen desaturation curve. Conversion to the deoxy state was achieved after the cell was flushed with nitrogen. These conditions were optimized for laboratory measurements and not as a model for circulatory studies. The data for native Hb were fit to the Adair equation using computation of an optimal nonlinear least-squares fit.
■
RESULTS AND DISCUSSION Self-Assembly of Hb-Biotin-Avidin-Biotin-Hb. As noted above, spontaneous association of oppositely charged globular proteins has been observed for other combinations.10,16 Solutions of Hb (pI 6.9) and avidin (pI 10.5) are transparent at 700 nm. Therefore, an increase in absorption at that wavelength indicates turbidity of the solution following a selfassembly process.10 For comparison, the combination of avidin with native Hb does not result in turbidity, while mixtures of cross-linked Hb with avidin produce notable changes in the character of the solutions (Figure 1). We determined the magnitude of the effect in buffers of various ionic strength and pH (Figure 2). In general, the aggregation is most apparent in buffers of low ionic strength. The effect is enhanced with a larger ratio of Hb to avidin (Figure 3) and by the addition of IHP, which associates with the polycationic site on Hb that binds 2,3-DPG (see Figure 4). As a control, we investigated if a similar response could be invoked using lysozyme (pI 11) in place of avidin. As with
Figure 3. Turbidity increase associated with an increase in the fumaryl cross-linked Hb to avidin ratio. Buffer is Tris, pH 9.0 (I = 1 mM). 2877
DOI: 10.1021/acs.biochem.6b00215 Biochemistry 2016, 55, 2875−2882
Article
Biochemistry
Figure 4. Turbidity changes associated with the addition of inositol hexaphosphate (IHP) to fumaryl cross-linked Hb/avidin mixtures in HEPES, pH 7.2 (I = 2 mM).
Figure 5. Absorbance at 700 nm, associated with solution turbidity, of protein mixtures of increasing lysozyme to native/cross-linked Hb ratio in Tris, pH 9.0 (I = 1 mM).
is produced as a result of the protein’s unfolding (Figure 7, bottom panel). Caccia et al.19 described the same fate for PEGmodified Hb, noting enhanced tetramer dissociation as a result of cysteine modification. Hb-Avidin Conjugation. In our initial approach, we sought to modify avidin with the biotin-maleimide reagent and then introduce Hb. However, this did not produce any of the desired higher molecular weight species. However, the alternative approach (the incubation of biotinylated Hb with avidin) gave the desired result (Scheme 2). Avidin’s biotin-binding sites are pairs located on opposing faces of the 67 kDa protein.9 Thus, we expect a maximum of two neighboring Hb tetramers per avidin. Excess biotinylated Hb in combination with avidin achieves saturation of the binding pockets. A single high molecular weight product was obtained, as deduced from size-exclusion HPLC (Figure 8, panel A). The product peak elutes much earlier than the ∼128 kDa Hb bis-tetramer reference material (Figure 8, panel B), which elutes at 32 min, suggesting a product of larger diameter. This is as expected for a conjugate with 2/1 Hb/avidin (∼195 kDa), as shown in Scheme 2. The peak at 40 min is consistent with the 32 kDa αβ-dimer derived from the biotinylated species that does not interact with avidin. When biotinylated Hb is combined with a large excess of avidin, an assembly with Hb/avidin in a 1/1 ratio (∼131 kDa) results (Figure 8, panel E). Scavenging of dissociating biotinylated dimers by avidin during their passage through the column can account for the absence of the 32 kDa peaks. These fragments were observed in native gel electrophoresis
Figure 6. Insoluble shards obtained upon glutaraldehyde treatment of a solution of native Hb and avidin.
(Figure 12, lane 2), confirming that the tetramer is intact in the assembly. An alternative explanation is that the direct interaction of the oppositely charged proteins stabilizes the tetramer against salt-induced dissociation. However, this is unlikely considering the importance of the cys-93 modification. Conjugates from fumaryl11 and trimesoyl20 cross-linked derivatives (Figure 8, panels C and D) were also prepared. Aggregation related to the propensity of these species to interact and/or weak avidin−biotin associations produced an 2878
DOI: 10.1021/acs.biochem.6b00215 Biochemistry 2016, 55, 2875−2882
Article
Biochemistry Scheme 1. Biotinylation of Hb with the Biotin-Maleimide Bifunctional Reagent
Figure 7. Spectral changes associated with native Hb (top panel) and native biotinylated Hb (bottom panel) being heated at 60 °C for 10 min. Figure 8. Size-exclusion HPLC traces of Hb-avidin conjugates with Hb bis-tetramer as a reference. Hb αβ-dimer (32 kDa, 40 min), Hb crosslinked tetramer (64 kDa, 36 min), avidin (67 kDa, 35 min), Hb bistetramer (128 kDa, 32 min), avidin +1 × Hb dimer (99 kDa, 30 min), avidin +2 × Hb dimer (131 kDa, 29 min). Aggregation >131 kDa (
