Self-Association of Mucin - Biomacromolecules (ACS Publications)

Biomacromolecules 2000, 1, 325-334

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Self-Association of Mucin Lev E. Bromberg*,† and David P. Barr‡ Department of Physics and Center for Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and Bruker Instruments, Inc., 19 Fortune Drive, Manning Park, Billerica, Massachusetts 01821 Received March 17, 2000

Aggregation phenomena in aqueous solutions of purified human tracheobronchial mucin have been studied by rheological methods, steady-state fluorescence, quasielastic light scattering, and spin probe techniques. At temperatures below 30 °C and concentrations above 15 mg/mL and in the absence of chaotropic agents, mucin solutions are viscoelastic gels. A gel-sol transition is observed at temperatures above 30 °C that is manifested by the diminishing storage modulus and a loss tangent above unity throughout the studied frequency range of the oscillatory shear. No decline in the mucin molecular weight is observed by sizeexclusion chromatography above 30 °C in the absence of redox agents or proteolytic enzymes. Aggregation of hydrophobic protein segments of the mucin chains at 37 °C is indicated by QELS experiments. The decreasing polarity of the microenvironment of pyrene solubilized into mucin solutions at temperatures above 30 °C, concomitant with the gel-sol transition, shows the hydrophobicity of the formed aggregates. ESR spectra of the fatty acid spin probe, 16-doxylstearic acid indicate that the aggregate-aqueous interface becomes more developed at elevated temperatures. Introduction Mucous glycoproteins (mucins) are large macromolecules secreted by various types of epithelial cells localized on the luminal surface of ductular and tubular structures. The backbone of mucins is a protein that is densely substituted with oligosaccharides, primarily via o-glycoside linkages to the serine and threonine residues. The length of the sidechain oligosaccharides varies from one to five sugar residues in mucins from submaxillary gland to up to 20 residues in tracheobronchial mucins.1-4 Disulfide bonds, comprising large (Mr 10-40 million Da) structures5,6 often link several glycoprotein chains (subunits) to each other. Within the mucin apoprotein backbone, domains with a very high density of oligosaccharide grafting alternate with “naked” domains devoid of saccharide residues.7,8 These “naked” protein segments can fold and unfold in response to changes in ionic strength,7 are susceptible to proteolytic attack, and are at least partly responsible for the interactions of mucins with enzymes, inorganic cations, fatty acids, albumins, and bacteria.9-12 Proteolytic digestion of “naked” protein domains usually leaves high-Mr glycopeptides (Tdomains).13 High molecular weight and intermolecular interactions lead to formation of gels, a prominent feature of all mucins. Physical gelation in aqueous mucin solutions is a well-documented phenomenon.4,14-20 It is in fact the biological function of gastric, cervical, and tracheobronchial mucins to form viscoelastic gels in order to protect cellular surfaces.21-23 The majority of the rheological studies of * To whom all correspondence should be addressed at 15 Sherwood Road, Swampscott, MA 01907. E-mail [email protected]. † Massachusetts Institute of Technology. ‡ Bruker Instruments, Inc.

mucins have been conducted in highly chaotropic media, such as 4-6 M guanidinium chloride.19,24 Such media disrupt the structure of water and ultimately prevent the entropydriven hydrophobic association between protein segments, as well as suppress ionic and hydrogen-bonding interactions. In the present study, we concentrated on the possible association of mucin chains due to intermolecular hydrophobic interactions. Therefore, experiments were conducted in the low ionic strength buffers in the absence of chaotropic agents. It appears that tracheobronchial mucin is prone to aggregation due to hydrophobic interactions between protein segments, as described below. Experimental Section Materials. Guanidine hydrochloride (GdHCl, 99+%), phenylmethylsulfonyl fluoride (PMSF, >99%), benzamidine hydrochloride hydrate (BaHCl, 98%), -amino-n-caproic acid (ACA, 99%), iodoacetamide (SigmaUltra), N-ethylmaleimide (SigmaUltra), deoxyribonuclease IV from bovine pancreas (DNase, ca. 2000 Kunitz units per mg), hyaluronidase from bovine testes (300-500 units per mg), bicinchoninic acid (BCA) kit for protein determination, type XIV bacterial protease from Streptomyces griseus, SDS-PAGE components, and Sepharose 2B (wet bead diameter 60-200 µm) were all obtained from Sigma and used as received. Pyrene (99%, optical grade), dithiothreitol (98%), and doxyl-16stearic acid were obtained from Aldrich Chemical Co. and were used as received except for pyrene, which was repeatedly recrystallized from absolute ethanol following sublimation. All other chemicals, gases, and organic solvents of the highest purity available were obtained from commercial sources.

10.1021/bm005532m CCC: $19.00 © 2000 American Chemical Society Published on Web 06/23/2000

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Table 1. Composition of Mucin component

wt %

amino acid

(mol %)

total protein fucose N-acetylglucosamine N-acetylgalactosamine galactose sialic acid sulfate

22.8 9.70 16.9 12.7 22.6 10.4 4.90

aspartic acid threonine serine glutamic acid proline glycine alanine valine methionine isoleucine leucine tyrosine phenylalanine histidine lysine arginine

4.68 23.4 11.4 5.75 13.6 7.70 9.93 4.98 0.68 1.93 5.93 0.78 1.52 2.31 2.23 3.18

Isolation and Characterization of Mucin. Mucous glycoprotein was prepared from tracheobronchial mucus secretions of two chronic-bronchitis patients as previously described.25 The mucus was then treated as described elsewhere,5,26-29 with certain variations to avoid the use of concentrated solutions of guanidinium chloride after the first step of mucus solubilization.4 In brief, the sputum samples (frozen immediately after collection) were thawed and dissolved while stirring in a 6 M GdHCl buffer solution containing 5 mM each of PMSF, BaHCl, ACA, iodoacetamide, and N-ethylmaleimide. The solution was then dialyzed against excess 0.02% sodium azide in deionized water at 4 °C for 48 h using Spectra/Por cellulose ester membrane (MW cutoff 10 kDa, Spectrum). DNase and hyaluronidase were added to remove DNA and proteoglycan contaminants, and the solution was centrifuged at 4 °C and 27000g for 1 h. The supernatant was concentrated by ultrafiltration using an Amicon Diaflow membrane (MW cutoff 100 kDa). After adjustment of the concentrate density to 1.4 g/mL with CsCl, the sample was subjected to density gradient ultracentrifugation using an Optima LE-80K ultracentrifuge (Beckman Instruments) equipped with an NVP-65 rotor. Two consecutive centrifugation runs (∼400000g, 4 °C, 24 h each) in CsCl yielded mucin fractions that were pooled, dialyzed using Spectra/Por cellulose ester membrane (MW cutoff 100 kDa, Spectrum) at 4 °C against excess 10 mM phosphate buffer/ 0.02% NaN3, and lyophilized. The mucin was stored at -70 °C prior to use. The unbound protein content of the obtained mucin was estimated to be below 0.1 wt % by using SDSPAGE (7.5% acrylamide, 0.1% SDS, silver staining). The lipid content was analyzed by the gas-liquid chromatography of the chloroform/methanol (2:1) extracts3,30 and was found to be negligible. The mucin was assayed for total carbohydrate content using the periodic acid-Schiff (PAS) reagent,31 for total protein content using the BCA assay,32 for sialic acid content by the resorcinol method,604 for monosaccharides by gas-liquid chromatography,34 for sulfate by the rhodizonate method,35 and for amino acid and amino sugar contents after HCl hydrolysis using a Hitachi L-8800 amino acid analyzer. Composition of the mucin is given in Table 1. It corresponds well with the reported composition of tracheobronchial mucins.2,3

Reduction and alkylation of purified mucin were performed as described elsewhere.3,36 Briefly, mucin was dissolved in 10 mM phosphate buffer/0.02% NaN3 at a final concentration of 0.3 mg/mL. Following deaeration of the solution by nitrogen bubbling for 0.5 h, dithiothreitol was added to result in 50 mM concentration. The solution was gently stirred in the dark for 1 or 6 h at ambient temperature, and iodoacetamide was subsequently added to yield a concentration of 0.15 M. After incubation in the dark for 1.5 h, the excess iodoacetamide was removed by the addition of dithiothreitol (final concentration 0.2 M). The reducedalkylated mucin species were dialyzed against excess deionized water and lyophilized. Proteolytic digestion of the purified mucin was carried out in 0.1 M phosphate buffer/0.02 NaN3 (pH 7.4) using a 1:10 w/w ratio of mucin to protease. The latter was added to the solution at the commencement of the incubation and after 24 h. The samples were incubated at 37 °C for 48 h, dialyzed against excess deionized water, and were subjected to centrifugation in CsCl gradient as described above. Chromatography. Gel filtration was conducted on a 30 × 4.5 cm column using Sepharose 2B preequilibrated with 6 M GdHCl, 10 mM sodium phosphate, and 0.02% NaN3 buffer and eluted with the same buffer. A 10 mL sample of mucin solution in 6 M GdHCl was loaded on the column, and 3 mL fractions were collected. Mucin concentration was measured by BCA assay. Size-exclusion chromatography (SEC) was ran at 15 °C on a Shimadzu LC-10A Series HPLC set up with a RID 10A refractive index detector. A 0.05 mg/mL sample of mucin dissolved in 6 M GdHCl solution was loaded onto a PL aquagel-OH 60 column (particle size 15 µm; dimensions 300 × 7.5 mm, Polymer Laboratories, Inc.) and then eluted using 6 M GdHCl and 10 mM phosphate buffer at 1 mL/min. The SEC system was calibrated in the MW range of 105-107 using poly(sodium acrylate) standards (American Polymer Standards Co.). Rheological Measurements. The mucin solutions were prepared by solubilizing lyophilized mucin specimen in 10 mM phosphate buffer/0.02% NaN3 at 4 °C under gentle stirring. The solution was filtered through a weighed Acrodisc nylon filter (Gelman Sciences) with pore diameter of 0.8 µm, centrifuged at 3000g for 5 min to remove air bubbles, and a sample (0.5 mL) was reequilibrated at the rheometer plate at 4 °C. Mucin concentration in the solutions was measured by BCA assay. Rheological measurements within angular frequency (ω) range of 0.0628 rad/s to 62.8 rad/s (minimum strain 6 × 10-5) were performed using a controlled stress Rheolyst Series AR1000 rheometer (TA Instruments, New Castle, DE) with a cone and plate geometry system (cone: diameter, 4 cm; angle, 2°; truncation, 57 µm) equipped with a solvent trap. Temperature control (internal resolution 0.016 °C) was provided by two Peltier plates. Fluorescence Measurements. Fluorescence spectra were recorded using a 10 mm path length quartz cell in a thermostated cuvette holder using a Shimadzu model RF5301 PC spectrofluorophotometer with a UV/vis polarizer under controlled temperature conditions (slit widths 1.5 nm) at right-angle geometry. Properties of the mucin solutions

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fitting a polynom up to the third order to the function ln(g(2)(t) - 1). The polynomial coefficients were converted into the coefficients of the cumulant expansion of the normalized autocorrelation function ln(g(1)(t)) ) ln(A) - Γt + µ2t2/2 - µ3t3/6

Figure 1. Representative emission spectra of pyrene in 10 mg/mL mucin solution in 10 mM phosphate buffer/0.02% NaN3 solution (pH 6.8) at two temperatures. I1 and I3 stand for the first and the third vibronic peak intensities. λex ) 335. Concentration of pyrene ) 0.6 µM. The spectra are normalized using the I1 band intensity.

were assessed by steady-state pyrene fluorescence studies. A stock solution of 1 mM pyrene in absolute methanol was prepared, from which 1-3 µL was added to 3.0 mL of an aerated aqueous sample. The sample was then allowed to equilibrate for 2-4 h, and emission (λex ) 335 nm) spectra were recorded. The ratio of the intensities of the third (384 nm) to the first (373 nm) vibronic peak (I3/I1) 37 in the emission spectra of the monomer pyrene was used to estimate the polarity of the pyrene microenvironment. Each spectrum was obtained by averaging three scans, corrected for scatter using equivalent blank glycoprotein solution, and I1/I3 values were averaged over three different estimates. No Fo¨rsterlike autofluorescence in the presence of pyrene was detected. The reproducibility of the results was better than 7%. Typical spectra are shown in Figure 1. The spectra were normalized using the I1 emission band. Quasi-Elastic Light Scattering (QELS). Mucin solutions were prepared by dissolving mucin in 10 mM phosphate buffer (0.3 mg/mL, pH 6.7) at 4 °C following filtration through a 0.8 µm Millipore filter into light-scattering cells, which were carefully sealed immediately after filtration. In a control experiment, the amount of mucin lost after filtration was found to be negligible by PAS assay. The cells were kept at 4 °C for 16 h, then were quickly placed into a holder at 10 or 37 °C, and the measurement commenced. The apparent hydrodynamic radii, RH, of mucin were measured in QELS experiments performed with a Brookhaven Instruments BI-200SM Goniometer, a BI-9000 Correlator, and a Spectra Physics He-Ne model 127 laser operating at a scattering angle of θ ) 90° and a wavelength of incident light of λ ) 632.8 nm at a power of 35 mW. The measured intensity autocorrelation function g(2)(t) was related to normalized autocorrelation function g(1)(t) by the equation g (t) ) B[1 + β|g (t)| ] (2)

(1)

where A is the amplitude and Γ is the relaxation rate related to the apparent diffusion coefficient D ) (Γ(q2)qf0. Here, q ) (4πn/λ) sin(θ/2) is the magnitude of the scattering vector, and n is the index of refraction. The apparent hydrodynamic radius RH is given by the Einstein-Stokes equation RH ) kBT/6πnD, where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. ESR Spectroscopy. Loading of glycoprotein solutions with spin probe doxyl-16-stearic acid (16-DSA) was achieved by adding solution of the 16-DSA in methanol to a 1 w/v% glycoprotein solution (pH 6.7). The resulting effective probe concentration of 0.19 mM allowing for optimum spectral resolution was found experimentally. ESR spectra were measured in quartz capillaries using a Bruker EMX 6/1 spectrometer operating at 9.65 GHz. A Bruker ER 4131-variable temperature accessory equipped with a ER 4102ST cavity was used to control the sample temperature. Spectra were analyzed using the WIN-EPR data processing program. The spectrometer settings for the ESR experiments were as follows: microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 2 G; center field, 3439 G; sweep width, 100 G; time constant, 82 ms; conversion time, 82 ms; sweep time, 84 s. The rotational correlation time was estimated from the equation: 37-40

τc ) 6.51 × 10-10∆H0

[( ) ( ) ] h0 h-1

0.5

+

h0 h+1

0.5

-2

where ∆H0 is the line width of the mid-field line (in gauss) and h-1, h0, and h+1 are the peak-to-peak heights of the low-, mid-, and high-field lines, respectively. We assume that the constant 6.51 × 10-10 can be used, to a good approximation, for our spin probes.41 The order parameter was calculated as S)

A|| - A⊥ Azz - (Axx + Ayy)/2

where Axx, Ayy, and Azz are the principal components of the A tensor in the absence of molecular motion and A|| and A⊥ are derived from the experimental spectra using maximum and minimum approximation.42 Order parameters were calculated using values Axx ) 6 G, Ayy ) 6 G, and Azz ) 32 G.43 Results and Discussion

2

where β and B are the instrumental constant and the baseline parameter, respectively. The RH values were found from the autocorrelation function using cumulant analysis, which was performed by

Rheological Study. Equilibration of purified mucin at 4 °C in 10 mM phosphate buffer/0.02% sodium azide solution (pH 6.8) in the concentration range 15-42 mg/mL yielded weak viscoelastic gels with a storage modulus (G′) exceeding the loss modulus (G′′) throughout the frequency range studied

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Figure 3. Retardation and recovery of 41.5 mg/mL mucin in 10 mM phosphate buffer/0.02% sodium azide (pH 6.8) preequilibrated at 4 or 37 °C for 24 h. Applied stress ) 0.2 Pa.

Figure 2. Frequency dependence of the storage (G′) and loss (G′′) moduli of the 15 mg/mL mucin solution in 10 mM phosphate buffer/ 0.02% sodium azide (pH 6.8) preequilibrated at 4 or 37 °C for 24 h. Conditions: oscillatory stress, 0.6 Pa; angular frequency, 6.28 rad/s. Open and filled points show G′′ and G′, respectively. The data points at 4 °C were fitted with the one-mode Maxwell model (solid lines) described by the following relations between the frequency-dependent storage (G′) and loss (G′′) moduli: G′(ω) ) Go$2τ2/(1 + $2τ2); G′′(ω) ) Go$τ/(1 + $2τ2); G′′($) ) [G′($)Go - G′($)2]0.5, where Go and τ are the plateau modulus and the terminal relaxation time, respectively.44

(Figure 2). A plateau on the G′ vs ω curve was observed that is characteristic of the quasiequilibrium modulus Go. In the low-frequency region (ω < 5 rad/s), the linear viscoelasticity of the mucin gels equilibrated at temperatures below 30° could be approximated as Maxwellian with a single relaxation time (Figure 2). Such behavior is typical of a Maxwell liquid such as solution of a polymer with hydrophobically interacting segments at concentrations slightly above gelation threshold.45-48 Mucin concentrations lower than 14.2 mg/mL did not yield gels with a clearly observable plateau modulus under conditions as in Figure 2, and the loss tangent (tan δ ) G′′/ G′) exceeded unity at any frequency. These results are in good agreement with observations by McCullagh et al. 4 who reported a sol-gel transition concentration of about 12 mg/ mL for high molecular weight tracheobronchial mucins dissolved in distilled water. At 37 °C the gel features of the mucin solutions were lost (Figure 2). A significant decrease of both G′ and G′′ was observed, with G′′ becoming higher than G′. The G′(ω) exhibited no plateau moduli. To compare rheological parameters of the mucin solutions in the sol and gel regimes, steady shear (creep) experiments were conducted. Figure 3 shows the outcome of the creep

Figure 4. Temperature dependency of the zero-shear viscosity of 37.6 mg/mL mucin in 10 mM phosphate buffer/0.02% sodium azide (pH 6.8) preequilibrated at a given temperature for 24 h.

measurements, carried out at two different temperatures with 41.5 mg/mL mucin in 10 mM phosphate buffer/0.02% sodium azide solution (pH 6.8). Under the experimental conditions, the solution preequilibrated at 37 °C was about 12-fold more compliant than the one at 4 °C and showed no signs of recovery upon cessation of the stress. The gel equilibrated at 4 °C recovered some of its strength, indicating reversibility of the physical cross-links. The creep experiments allow estimation of the zero shear viscosity (ηo) according to:49 γ(t)/σo ) J0e + t/η0 where t is the time, γ(t) is the shear strain, σo is the shear stress, and J 0e is the steady-state shear compliance. The zero-shear viscosity of the mucin solution in the absence of chaotropic agents was temperature-dependent, showing an abrupt decrease above 30 °C (Figure 4). This result, along with the oscillatory shear experiments, point to the gel-sol transitions caused by the elevated temperature. It might be hypothesized that this observation is due to some deleterious processes in the secondary structure of mucin, since analogous gel-sol transitions have been observed upon reduction of disulfide bonds that connect several glycoprotein chains (subunits) into a mucus gel.16


Figure 5. Elution profile of purified mucin (A), mucin reduced for 1 h by dithiothreitol (B), and mucin equilibrated in 10 mM phosphate buffer/0.02% NaN3 at 37 °C for 24 h (C). After treatment, each mucin species was lyophilized and redissolved in 6 M GdHCl/0.02% NaN3/ 10 mM phosphate buffer. Arrow shows elution of blue dextran (average MW 5 × 106). For other experimental details, see text.

Figure 6. Size-exclusion chromatograms of purified mucin (A) and the same fraction kept at 37 °C in 10 mM phosphate buffer/0.02 NaN3 for 24 h (B). After treatment, each mucin species was lyophilized and redissolved in 6 M GdHCl/0.02% NaN3/10 mM phosphate buffer. For other details, see Experimental section.

Redox agents such as mercaptoethanol usually aid such reduction. Alternatively, a proteolysis leading to a chain scission of the protein segments (and therefore to a destruction of the gel) may occur by contaminant proteolytic enzymes.16 To ascertain the mechanism of the observed gelliquid transition, a series of chromatographic experiments was conducted. Somewhat unexpectedly, using the FPLC method described in the Experimental Section, we did not detect any significant changes in the content of native (oxidized) mucin even after 24 h-long exposure to 37 °C of the mucin solution in 10 mM phosphate buffer/0.02% NaN3 (pH 6.8) (Figure 5). To evaluate changes, if any, in the molecular weight, a fraction of the mucin kept at 37 °C for 24 h was snap-frozen in liquid nitrogen and lyophilized. The fraction was redissolved in 6 M guanidinium chloride/0.02% NaN3 and subjected to size-exclusion chromatography as described in Experimental Section. As can be seen from Figure 6, no significant reduction in molecular weight of the mucin was observed as a result of exposure to 37 °C under these conditions. Hence, liquefaction of the viscoelastic gels in our case cannot be attributed to the lessening molecular weight due to the redox or proteolytic reactions. Significant


reduction of the disulfide bonds can also be ruled out. Instead, such changes can be attributed to a phase separation leading to dense domains interspersed with water-rich loose domains. The latter would alter the rheology of the mucin. The gel rheology is a signature of connectivity. The local clusters in gels are interconnected into a supermolecular structure. The connectivity can be achieved through an immediate contact between structural units, a network of bridging molecules possessing segments in neighboring crystalline phases, or via an impingement of amorphous chains, immobilized by their attachment to segments within a crystalline structure, with similarly immobilized chains from adjacent domains.50 The widely accepted concept of formation of weak mucin gels due to the interdigitation of the closely packed, side-chain oligosaccharides grafted onto protein backbone during the mucus development4,51 would be analogous to the case of bridging chains with segments in neighboring crystalline phases. In our case, the phase separation process can be followed by a gel-sol transition with a decrease in G′ and viscosity when the initially dissolved viscoelastic mucin-rich component separates from the water-rich matrix. Such phase-separation phenomena are well-known in polymer blends.52,53 The interdigitation of the oligosaccharide chains must be altered upon this phase separation. The formation of microphase-separated gels in aqueous solution of the human tracheobronchial mucin has been suggested in the literature,24 and pH-induced reversible sol-gel transitions of gastric mucin have also been documented.54 However, the temperature-induced gel-sol transitions do not seem to be known, possibly because most studies of the macromolecular structure of mucins have been conducted in chaotropic media. We have attempted to shed light on the properties of aggregates resulting from phase separation using QELS, fluorometry, and ESR techniques described next. Light-Scattering Study. The time-dependent hydrodynamic radii of the whole mucin, its reduced subunits and T-domains are shown in Figure 7. Aggregation of mucin and its subunits were quite pronounced at 37 °C, resulting in a 1.7-fold increase in the RH over 2 h. Initial RH values of about 200, 70, and 30 nm found for the native mucin, its reduced subunits, and the domains devoid of protein segments, respectively, correspond well with the reported radii of tracheobronchial2,55 and cervical27,28 mucins. An increase in RH at 10 °C was not observed. Importantly, no aggregation of T-domains devoid of protein was apparent (Figure 7). This observation provides a strong evidence for associative hydrophobic interactions arising among protein domains belonging to different mucin chains. An analogy can be drawn between these intermolecular associations and the entropy-driven assembly of hydrophobic segments of the hydrophobically modified polyelectrolytes. 56 Steady-State Fluorescence Study. The fluorescence intensity ratio between the first and third vibronic peaks (I1/ I3) of excited-state monomeric pyrene emission was determined over a range of mucin concentrations in solutions of 10 mM phosphate buffer/0.02% NaN3 at different temperatures (Figures 8 and 9). The pronounced change in the characteristic I1/I3 ratio with temperature above 30 °C (Figure

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Figure 9. Intensity ratio (I1/I3) for pyrene in mucin solution in 10 mM phosphate buffer/0.02% NaN3 (pH 6.8) as a function of protein concentration. Filled and open points show data obtained at 20 and 37 °C, respectively. Pyrene concentration is 0.6 µM throughout. Protein concentration in mucin solution is measured by BCA assay.

Figure 7. Hydrodynamic radii of the whole mucin (A) and mucin subunits and T-domains (B). Mucin subunits and T-domains are obtained by reduction/alkylation and proteolytic digestion, respectively, of the whole mucin, as described in Experimental Section. Squares and circles indicate data obtained at 37 and 10 °C, respectively. In B, open points show data obtained for mucin subunits, whereas filled points indicate data for T-domains.

Figure 8. Temperature dependency of the ratio of the first to the third vibronic peak intensities (I1/I3) of the emission spectra of monomeric pyrene solubilized into mucin solutions in 10 mM phosphate buffer/0.02% NaN3 (pH 6.8). Open and filled points indicate data for 11.2 and 41.5 mg/mL mucin solutions, respectively.

8) indicates that the mucin aggregates formed as a result of concomitant collapse of the gel structure (compare with Figures 4 and 7) provide hydrophobic environment for the pyrene. The variations in the vibrational fine structure of

pyrene monomer emission has been widely used as a spectroscopic tool to study aggregate properties.37,56,57 The I3/I1 value is a useful indicator of the local dielectric constant surrounding the probe58 and has values of approximately 1.9 in water, 1.1-1.4 in common short-chain alcohols, 0.6-0.7 in hydrocarbon media, and 0.7-0.9 in micelles of common surfactants and amphiphilic polymer systems.37,59 As can be seen, the I1/I3 values in 41.5 mg/mL mucin solution at T >30 °C became comparable to the ones in hydrocarbons. To ascertain the nature of the hydrophobes in mucin, the concentration of mucin in 10 mM phosphate/0.02% NaN3 was varied, and the I1/I3 value and the total protein concentration in the resulting solutions were measured. Even concentrations of mucin as low as 0.1-0.2 mg/mL lead to a decrease of the I1/I3 from the value (1.87) in water59 to about 1.65, indicating the presence of the hydrophobic components. As concentration of mucous protein increases above 0.1 mg/ mL, the I1/I3 starts to decrease steeply, indicating the onset of aggregation (compare with Figure 7). The shape of the I1/I3 vs protein concentration (Cp) isotherm allows for estimate of the effective partition coefficient of pyrene between protein segments and aqueous phase (Figures 9 and 10).60,61 A plot of vs Cp is a straight line (R2 > 0.96) both at 20 and 37 °C with a slope proportional to K (Figure 10). Approximating F by 1.0 g/mL and taking f ≈ 1, 62 the slopes in Figure 10 yield K ≈ 1500 and 3400 for 20 and 37 °C, respectively. These values are much smaller than K on the order of 105 typically observed in solutions of micelle-forming hydrophobically modified polyelectrolytes,37,63 suggesting that hydrophobic domains in mucin are much smaller. However, the increase in K, as well as the QELS results (Figure 7), indicates that aggregates formed at elevated temperature are capable of accommodating a larger number of pyrene molecules. Spin Probe Studies. Since hydrophobic interactions are a major contributor in the binding energy of fatty acids to proteins,64,65 we rationalized that the aforementioned hydrophobic associations in mucin could be studied using ESR in


Figure 10. Partitioning of pyrene between protein segments of mucin and aqueous phases as a function of protein concentration at pH 6.8. See text for details.

conjunction with a fatty acid spin probe. The 16-doxylstearic acid (16-DSA) spin probe was applied to detect changes in 10 mg/mL mucin gels (pH 6.7) with increasing temperature (Figure 11). In the 16-DSA, the nitroxyl label is located farther away from the carboxyl group than in other frequently used nitroxyl-labeled stearic acids with n ) 5, 7, or 12. The 16-DSA is less prone to micellization than 5- or 7-DSA.64 Hence the 16-DSA is an effective probe of the depths of lipid bilayers, protein aggregates, and other hydrophobic domains.40,64,66-68 As can be seen from Figure 11, the spectra of the probe significantly changed above 20 °C evolving into a more


pronounced triplet characteristic of a nitroxyl solubilized at higher concentration. Relative concentration of the probe in the mucin solution as expressed through the double integrated intensity of the M ) -1 nitrogen hyperfine line69 steeply increased above 20 °C to arrive to near-equilibrium values at 37 °C (Figure 12). The composite structure of the spectra measured at lower temperatures hinted at the presence of a slower and a faster component. The slow component can be attributed to the dissociated, anionic form of the probe, whereas the fast spectrum has been attributed to the undissociated, less polar species of the doxylstearic acid.40,70,71 The undissociated species tends to bind to nonpolar domains, such as the interior of the aggregates in the collapsed gels or within hydrophobic cores of micelles of the polymeric surfactants.40 As the probe became more solubilized at increased temperatures, a triplet or doublets were more apparent in the spectrum, which is due to the coupling of the electron to the natural abundance of 13C (i.e., spin (1/2).72 A dramatic increase in relative concentration of the probe (Figure 12) can be attributed to the decreasing polarity of the probe’s microenvironment. This conclusion is supported by the decline in the order parameter S with temperature (Figure 13). The decrease of the S values and associated hyperfine splitting (estimated from the extreme separations of the outer peaks) with temperature in the spectra of doxylfatty acids reflect the decreasing local polarity.42,73 Such changes are typically associated with the doxyl group diffusing deeper into the hydrocarbon environment of phospholipid membranes or hydrophobic pockets of proteins.42,74

Figure 11. ESR spectra of 16-DSA in 10 mg/mL mucin aqueous solution (pH 6.7) at varying temperatures. Inset shows the method of parameter calculation.

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reduced and the probe is allowed to rotate more freely. Thus, the decrease in the microviscosity (η) of the probe’s environment with temperature.76 These EPR observations are consistent with the hypothesis of a temperature-induced hydrophobic aggregation in mucin solutions proposed above on the basis of rheological, QELS, and fluorometric experiments. Conclusions

Figure 12. Temperature dependence of the relative concentration of the 16-DSA spin probe solubilized in 10 mg/mL aqueous mucin solution. Relative concentration of the probe is expressed as [∆H-12h(-1)]T/[∆H-12h(-1)]10 °C where h(-1) is the peak height and ∆H-1 is the peak-to-peak width of the -1 line.69

Figure 13. Temperature dependence of the order parameter (S, filled points) and rotational correlation time (τc, open points) of the 16-DSA spin probe in 10 mg/mL aqueous mucin solution.

Slight increase of the nitrogen hyperfine coupling constant of the radical (aN) from 15.6 to about 15.9 G was observed in temperature ranges below 20 and 30-40 °C, respectively. Interestingly, opposite trends (i.e., increase of S and decrease of aN with temperature) are observed when a probe is buried within hydrophobic microenvironment such as cores of the polymeric micelles.37 On the contrary, trends analogous to the ones reported herein were observed for the 5-doxylstearic acid located mostly near the polar interface between a hydrophobic domain and aqueous environment.40,75 More restricted mobility of the probe near the interface results in the increase of the aN with a growth of the interfacial surface upon aggregation. There, the partially dissociated carboxyl group would be close to the hydrated domains, while the hydrocarbon tail of the probe (and thus the doxyl) would be located near the hydrophobic domain. Association of the 16doxylstearic acid with the hydrated polar regions bordering the nonpolar mucin aggregates is further supported by the decrease of the rotational correlation time from 27 × 10-10 to about 1.4 × 10-10 s at 10 and 30-50 °C, respectively (Figure 12). It may be suggested that upon heating, the hydrated regions lose hydration layers where the carboxyl group of the probe is located, anchoring the probe. Without such anchoring, the restriction of the probe mobility is

The present study dealt with aggregation phenomena in aqueous solutions of the mucous glycoprotein (mucin) prepared from tracheobronchial mucus secretions. In the absence of chaotropic agents, a gel-sol transition is observed at temperatures above 30 °C and mucin concentrations above 14 mg/mL. The transition is manifested by the loss of plateau storage modulus and dramatic decline of both storage and loss moduli, with the loss tangent becoming above unity throughout the studied frequency range of the oscillatory shear. Chromatographic measurements do not indicate significant changes in the molecular weight of the mucin due to deleterious processes at elevated temperature, which might otherwise be invoked to explain the collapse of the gel features. Instead, a microphase separation caused by intermolecular hydrophobic interactions among protein segments of mucin seems to be a plausible cause of the gel-sol transition. This hypothesis is supported by the QELS observation of the time-dependent increase in hydrodynamic radius of mucin chains and subunits at 37 °C, but not at 10 °C. Since such aggregation is absent in the mucin species stripped of the protein segments, these results confirm the notion of aggregation due to associative hydrophobic interactions among protein domains. The decrease of the I1/I3 value of the emission spectra of pyrene solubilized into mucin solutions at temperatures above 30 °C, concomitant with the gel-sol transition, shows that the formed aggregates are hydrophobic in nature. Temperature-induced solubilization of the fatty acid spin probe, 16-doxylstearic acid (16-DSA) into the mucin is attributed to the decreasing polarity of the 16-DSA microenvironment, as indicated by the decrease in the order parameter hyperfine splitting constant. Temperature-induced decline of the rotational correlation time and increase of the nitrogen hyperfine coupling constant indicate that the 16-DSA is located near the polar interface between a hydrophobic domain and aqueous environment, which becomes more prominent at elevated temperature. This results is in agreement with observations of the binding of 16-DSA to proteins, where the probe protrudes from the hydrophobic domain of the protein and resides in a more polar and less hindered environment, which permits a significant degree of internal motion.64 Tendency of tracheobronchial mucins to aggregate in the absence of high amounts of salt has been noted before.4,77,78 Electron microscopy shows that on aggregation, tracheobronchial mucin forms an interwoven network with apparent dense core-like domains with some associated filamentous structures.79 The present study points out to the interchain hydrophobic interactions as a primary cause of such aggregation. It would appear that mucus layer secreted by


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(75) Bromberg, L. E.; Goldfeld, M. G. Polym. Prepr. 1998, 39, 681. (76) The Debye-Stokes-Einstein equation τc ) 4πηr3/3kT, where r is the radius of the tumbling entity (spin probe), and k and T are the Boltzmann constant and absolute temperature, respectively. (77) Chase, K. V.; Naziruddin, B.; Desai, V. C.; Flux, M.; Sachdev, G. P. Exp. Lung Res. 1989, 15, 721.

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Self-Association of Mucin - Biomacromolecules (ACS Publications)

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