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Morphology and Molecular Mobility of Fibrous Hard r-Keratins by 1H, 13C, and 129
Xe
NMR
Maria Baias,† Dan E. Demco,*,‡,§ Daniel Istrate,‡ Crisan Popescu,*,‡,| Bernhard Blümich,† and
Martin Möller‡
DWI an der RWTH Aachen, Pauwelsstrasse 8, D-52056 Aachen, Germany, Institut für Technische und
Makromolekulare Chemie, RWTH Aachen UniVersity, Worringer Weg 1, D-52074 Aachen, Germany, Technical
UniVersity Cluj-Napoca, Department of Physics, RO-400020 Cluj-Napoca, Romania, and UniVersity “Aurel
Vlaicu” Arad, Bd. ReVolutiei 77, RO-310130 Arad, Romania
ReceiVed: May 14, 2009; ReVised Manuscript ReceiVed: July 14, 2009
The morphology and molecular mobility changes of the side chains for hard R-keratin due to oxidative and
reductive/oxidative treatments for temperatures around the DSC denaturation peak were investigated by 1H,
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13
C, and 129Xe NMR spectroscopy and 1H spin diffusion. Proton wide-line spectra were used to obtain the
phase composition (rigid, interface, and amorphous fractions) and molecular dynamics of each phase. Proton
spin diffusion experiments using a double-quantum filter and initial rate approximation were employed to
obtain the dependence of the rigid domain sizes on chemical treatments and denaturation temperatures. A
drastic reduction in the rigid domain thickness takes place for the reductive/oxidative treatment. The keratin
Publication Date (Web): August 6, 2009 | doi: 10.1021/jp904484r
mobility gradient in the interfacial region at different denaturation temperatures was measured for hard R-keratin
from 1H spin diffusion data. 13C CPMAS spectra were used to provide a detailed examination of the effects
of the chemical treatments especially on the disulfide bonds. Thermally polarized 129Xe spectra suggest
the existence of voids in the hard R-keratin induced by the reductive and oxidative treatment. The surface
of the hard R-keratin fiber surface is probed by the laser hyperpolarized 129Xe. A qualitative model describing
the changes induced in hard R-keratin protein by chemical transformation was developed and could be correlated
with the changes in domain thickness, phase composition, and molecular dynamics.
1. Introduction Although the above model was proposed for describing the
The hard R-keratin is a filament protein found in mammalian mechanic behavior of keratins, it appeared to also be suitable
epidermal appendages (hairs, quills, horn, nails, etc.) distinct for explaining the high denaturation temperature found in
from feather β-keratin found in avian and reptilian tissues. The keratins.8,9 In soluble proteins, the helix denaturates (unfolds)
hair is the most sophisticated biological composite material.1 at temperatures up to 80 °C. There are no data on the
The structure of hard R-keratin is characterized by three denaturation temperature of IFs alone (not surrounded by a
structural hierarchy levels.2 At high resolution, the intermediate matrix), but one may expect that the R-helix from keratins would
filament (IF) protein is made of a central rod domain of amino also unfold at temperatures around 80 °C. It is assumed that
acid sequences (1A, 1B, 2A, and 2B) containing an aminoacid the fact that keratin proteins show denaturation at above 100
heptad repeat unit, and separated by loop links (L1, L12, and °C is due to the rigidity of the matrix, whose viscosity impedes
L2).3,4 At the extremity of the rod domain are located the the unfolding of the helix. The viscosity (and cross-link) of the
globular C- and N-terminal domains arranged mostly in β-sheets matrix governs, therefore, the segmental mobility of the R-helix
and formed of sulfur rich compounds.5,6 Two strands of and the unfolding reaction (denaturation).
R-helices are coiled coil to form a superhelical dimer. At the A similar model was proposed for collagen based materials.10
medium resolution, i.e., the intermediate level arrangement of The model suggests that adding solvents able to decrease the
the heterodimers inside IFs, the molecules are assembled both viscosity of the matrix depresses the temperature of unfolding.
longitudinally and laterally in an ensemble called a microfibril.7 This has been indeed noticed in differential scanning calorimetry
The dimers are associated as tetramers, which group to form a (DSC) experiments with keratin fibers11,12 and with collagen
long cylinder-shaped intermediate filament with 32 keratin based materials (parchments, leathers) in a water environment.13-15
chains in cross section. At lower structural resolution, the Understanding properly how the keratins protect the intermediate
bundles of parallel IFs are organized in an amorphous and filaments against thermal denaturation until high values of
disordered crystalline lateral network. These are embedded in temperature is of a clear interest for the fundamental knowledge
a sulfur-rich protein matrix of intermediate filament associated of protein denaturation. The role of the matrix in this process
proteins (IFAPs) and form a macrofibril, the main morphological may suggest ways for designing high-temperature stable proteins
component of hard R-keratin fibers.1,2 as new biomaterials.
Multinuclear and multidimensional liquid- and solid-state
* Corresponding authors. Fax: +49-241-233-01. E-mail: demco@ NMR are important techniques in structural biology.16-18
mc.rwth-aachen.de (D.E.D.); cpopescu@dwi.rwth-aachen.de (C.P.).
†
Recently, a 13C and 2H solid-state NMR study of an R-keratin
RWTH Aachen University. sourced from equine hoof has revealed a strong dependence of
‡
DWI an der RWTH Aachen.
§
Technical University Cluj-Napoca. molecular conformation and molecular dynamics on the degree
|
University “Aurel Vlaicu” Arad. of hydration of the material.19 In particular, dehydration results
10.1021/jp904484r CCC: $40.75 2009 American Chemical Society
Published on Web 08/06/2009
Morphology and Molecular Mobility of Hard R-Keratins J. Phys. Chem. B, Vol. 113, No. 35, 2009 12137
in a much more rigid and ordered structure, with a loss of with ammonia. The fibers were covered with the paste, massaged
R-helical components in the structure and breaking of cysteine gently between the fingers, and left 30 min to react at room
disulfide bonds. Moreover, the molecular dynamics and struc- temperature. The fibers were then rinsed thoroughly until the
tural organization of mouse epidermal keratin intermediate pH of the aqueous extract was checked to be 7. The treatment
filaments (IFs) have been studied via 13C and 2H spectroscopy was resumed two more times.
and relaxometry on IFs labeled with isotopically enriched amino The reductive/oxidative treatment was performed with thiogly-
acids.20 Solid-state 31P NMR spectroscopy was also applied to colic acid (TGA) and hydrogen peroxide. A 1 g portion of
the analysis of phosphorylated wool keratin to investigate the keratin fibers, prewetted with water, was immersed for 30 s in
changes induced on the surface of wool keratin.21 the reducing solution of 8% w/w TGA at pH 8.5-9 adjusted
The clarification of the fine structure of fibrous proteins like with ammonia, the solution excess being removed by gently
keratin in the solid state is important for the understanding of pressing the fibers between the fingers, then covered with plastic
their nature. This may be achieved because 13C and 15N chemical folia and let to react for 30 min at room temperature. The fibers
shifts of polypeptides are substantially dependent on their main- were then rinsed with tap water (3 min) and immersed in the
chain conformations such as R-helix and β-sheet forms. Using
oxidative (hydrogen peroxide 3%) solution adjusted at pH 4.5
this method, it was confirmed that both right-handed R-helix
with phosphoric acid, for 30 s. After squeezing between fingers,
and β-sheet forms exist in native wool fiber.22-24
fibers were allowed to react with the oxidative solution for 10
Spin diffusion NMR was proved to be a useful method for
min, at room temperature. Finally, the fibers were washed
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For the interpretation of these experiments, the solutions of the bonds were broken and protected by alkylation. A 1 g portion
spin diffusion equations for two-dimensional square and cylin- of fibers was reacted with 8 mL of 0.5 M tris(2-carboxyeth-
drical morphologies were employed. The keratin mobility yl)phosphine hydrochloride (TCEP), at pH 7 adjusted with
gradient in the interfacial region at different denaturation ammonium hydroxide, 48 h under continuous stirring at room
temperatures was also measured from the 1H spin diffusion data. temperature. After removing the TCEP solution excess, 10 mL
A qualitative model describing the denaturation process of of iodacetamide (1 M, pH 8) was added without previously
hydrated keratin protein was developed that explains the changes washing the fiber material (to avoid reformation of disulfide
in domain thickness, spin diffusivities, phase composition, and bonds) and the sample was kept for 48 h under continuous
thermodynamic parameters. stirring at room temperature and in the dark. Eventually, the
The aim of this work is to investigate the changes induced fibers were rinsed under tap water for 3 min, two times
by various chemical treatments on hair keratin and the thermal subsequently washed with a solution of Texapon N70, 0.1 mL/L
denaturation process of these materials by 1H NMR wide-line (70% natrium laurethsulfat), rinsed again with warm water for
spectroscopy and 1H spin diffusion. Moreover, cross-polarization 1 min and then tap water for 3 min, and dried in air.
magic angle spinning (CPMAS) 13C NMR spectra and thermally 2.2. DSC Measurements. The DSC experiments were run
polarized and hyperpolarized 129Xe spectra were used for this in a DSC-7 Perkin-Elmer instrument calibrated with indium and
purpose. The phase (fraction) composition is measured from palmitic acid, both of high purity, using pressure resistant
1
H wide-line spectra ex situ for native and chemically treated stainless steel large volume capsules. DSC calibration was done
Caucasian hair sampled at different temperatures during the with indium and palmitic acid, both of high purity. We used a
denaturation process. Three fractions are detected, i.e., rigid, heating rate of 10 K/min for temperatures ranging from 60 to
interfacial, and amorphous. The rigid domain sizes for the hair 180 °C. Each experiment was repeated three to five times, for
samples were measured by 1H spin diffusion using initial-rate ensuring the reproducibility of data.
approximation. The molecular dynamics gradient of the inter- Prior to the DSC measurements, the samples were cut into
facial region was investigated using the 1H spin diffusion NMR fine snippets (about 2 mm) and stored under controlled
experiments. The changes in the degree of fibrous hard R-keratin conditions (about 24 h at 22 °C and 55% relative humidity) to
organization, the amount of different phases, and molecular
ensure invariant water contents. The amount of 7-10 mg of
dynamics are discussed in correlation with the type of hair
each sample snippets were weighted and placed in crucible for
chemical treatments and temperatures during the thermal
the DSC measurements. Prior to sealing a crucible, 50 µL of
denaturation process.
distilled water (pH 6.7) was added, and the sealed crucible was
stored overnight for about 14 h, to allow the fibers to wet. The
2. Experimental Section samples for NMR measurements were gathered from DSC
2.1. Samples. The hard R-keratin fibers used for this study experiments by taking the pans at various moments linked to
were of Caucasian dark-brown hair, supplied by Kerling thermal events as disclosed by DSC. Three different samples,
International Haarfabrik GmbH, Germany. The fibers were which will be reported below, were collected this way at various
cleaned with 1% lauryl ether sulfate (LES) and dried at room temperatures including the denaturation temperature.
temperature prior to working with them. The pH of their aqueous 2.3. Proton and 13C NMR Measurements. Proton solid-
extract was found to be 6.5-7. state NMR spectra, 1H double-quantum (DQ) build-up curves,
1
The damage of the keratin fibers was induced by oxidative H spin diffusion, and 13C CPMAS spectra were measured on
and reductive/oxidative chemical treatments, respectively. a Bruker DSX-500 spectrometer operating at 500.45 and 125.84
The oxidative treatment was performed on 1 g of keratin fibers MHz for 1H and 13C, respectively. Proton NMR data were
with 0.2 g of potassium persulphate mixed with 1.2 mL of 6% collected at room temperature for nonspinning samples. The
hydrogen peroxide solution to form a paste adjusted at pH 8.5-9 dead time of the spectrometer is 5.5 µs. The length of a π/2
12138 J. Phys. Chem. B, Vol. 113, No. 35, 2009 Baias et al.
pulse was about 5.5 µs, the dwell time was 2 µs, and the recycle
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Mz(b,
r t) A highly efficient dipolar filter is characterized by the initial
m(b,
r t) ) (1) concentration of magnetization: mR0 * 0 and mM0 ) 0. For such
F(b)∆V(
r b)
r
a condition, eq 4 has the form
where Mz(r b,t) is the total z-magnetization and ∆V(r b) is the
infinitesimal volume around the point defined by the vector b. r FR√DRmR0 FM√DMmR0
The number density of spins is denoted by F(r b). mR(b,
r t) ) + ×
FR√DR + FM√DM FR√DR + FM√DM
In the limit of isotropic spin diffusion and spatially constant
{ dR /2 - xi
}
ε
spin diffusivity (D), the spin diffusion equation has the form
∏ erf (6)
∂m(b,
r t)
i)1 √4DRt
) D∇2m(b,
r t) (2)
∂t
Using the results presented in ref 27 (eq 30) and the above eqs
3, 5, and 6, we get for the time evolution of the integral intensity
The instantaneous NMR observables in a spin diffusion of the NMR signal from domain R the relationship
experiment are represented by the normalized integral intensity
Ii(t)/I0 of the ith component of the NMR spectrum with the total
integral intensity I0. More specific, the NMR spin diffusion IR(t) FR√DR FM√DM
) + ×
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{ 4√DRt 1
[ ( )]}
ε
dR
1- - ierfc (7)
dR √π 4√DRt
Publication Date (Web): August 6, 2009 | doi: 10.1021/jp904484r
{ }
fraction in the following considerations.
The solution of the spin diffusion equation for the composite 4ε√DRt
1- (9)
medium of finite source and semi-infinite sink can be obtained √πdR
using the solution for a one-dimensional (1D) composite
medium.27,31-33 For an ε-dimensional diffusion process with ε
> 1, the solution of the spin diffusion equation can be written The spin diffusion decay curve described by eq 9 corresponds
simply as a product of the solutions for the 1D diffusion to an initial slope straight line that intersects the (t)1/2 axis at
process.27 For this, the essential condition is that the initial (t0)1/2. The domain thickness dR for a rectangular 1D, 2D, or
conditions must be expressible as a product of those for the 3D morphology is given from eq 9 by
one-variable problems taken separately. The space and time
evolution of the concentration of magnetization in the R domain FM√DRDM
4ε
is given by27 dR ≈ √t0 (10)
√π FR√DR + FM√DM
FR√DRmR0 + FM√DMmM0
mR(b,
r t) ) - In the time regime in which the spin diffusion is not affected
FR√DR + FM√DM by the spin-lattice relaxation, the theorem of total magnetization
FM√DM(mM0 - mR0)
{ }
ε
dR /2 - xi conservation leads to
FR√DR + FM√DM
∏ erf (4)
i)1 √4DRt IR(t) IM(t)
+ )1 (11)
I0 I0
where xi are the coordinates of the vector b(x
r 1, x2, x3) and xi <
dR/2. The error function is defined as
Hence, from eqs 9 and 11, the time evolution of the spin
diffusion build-up curve for the sink domain (M) has the same
2
∫0z e-x
2
erf(z) ) dx (5) slope as that of the source domain. The intercept of the tangent
√π straight line starting from t ) 0 with the horizontal line at I(0)/
12140 J. Phys. Chem. B, Vol. 113, No. 35, 2009 Baias et al.
Figure 3. DSC signals of hard R-keratin, in the native state and after
oxidative and reductive/oxidative treatments in D2O for the denaturation
temperatures. The arrows mark the temperatures at which the samples
were used in the NMR measurements. Figure 4. Proton wide-line NMR spectrum of hard R-keratin. All NMR
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morphology is a good approximation for the morphology with reveal a slight increase in the relative amount of rigid phase
both finite domains in the initial-rate regime. The thickness of and a decrease of the interface for native hard R-keratin. We
the mobile domain dM can be obtained by the same procedure can note an opposite behavior for the sample after the oxida-
discussed above using a Goldman-Shen dipolar filter.34 tive and reductive/oxidative treatments. The amount of mobile
We can also note that the derivation of the relationship for (amorphous) fraction is not essentially affected by the dena-
dR (eq 10) employs only the solution of the spin diffusion turation temperature. The reductive/oxidative treatment increases
equation with corresponding initial and boundary conditions. the relative amount of rigid fraction as the expense of interface
Moreover, the time evolution is considered for the normalized compared to the hard R-keratin.
magnetization. This is not the case for the intercept spin The molecular dynamics of hard R-keratin, after oxidative
diffusion time reported in refs 26 and 35 where the phase and reductive and oxidative treatments for the temperature range
structure considerations and magnetization at equilibrium were where denaturation occurs reflected in the line width of the 1H
taken into account. spectral components is shown for the rigid, interface, and mobile
fractions in Figure 6. In general, denaturation at 180 °C induces
4. Results and Discussion a greater disorganization in the nanostructured keratin and hence
4.1. Thermal Denaturation by DSC. Typical DSC plots in a large molecular mobility. An exception is the mobile fraction
D2O for the temperature range of denaturation of hard R-keratin of the sample after reductive/oxidative treatment. The molecular
in the untreated state, after oxidative, and after reductive and motion is strongly hindered by the matrix disorganization
oxidative treatment, respectively, are shown in Figure 3. The induced by this chemical treatment. The molecular motions are
endothermal process recorded around 154 °C for the untreated more hindered for the rigid phase and interface of hard R-keratin.
sample is attributed to the thermal denaturation of keratin by 4.3. Double-Quantum Dipolar Filter for 1H Spin Diffu-
melting of the R-helix crystalline structure.26 The scenario for sion. The spin diffusion experiments observe the equilibration
the thermal denaturation of hard R-keratin in the native state of spatially heterogeneous magnetization over the sample. A
and after chemical treatments as reflected in the DSC and NMR magnetization gradient can be created, for example, with a
data will be discussed below. dipolar filter which excites double-quantum (DQ) coherences.24
4.2. Proton NMR Spectra, Phase Composition, and Mo- This type of filter is more advantageous than a dipolar filter for
lecular Dynamics. The proton NMR spectrum of hard R-keratin, mobile domains because it allows a more accurate detection of
recorded under static conditions at room temperature, is the narrow signals on the top of the broad component as
presented in Figure 4. The best fitting parameters have been compared to the detection of a broad component under a narrow
found by decomposing the spectra in three lines described by a signal. This is valid especially at short diffusion times when
Gaussian, a Lorenzian, and a combination of Gaussian and the magnetization of one of the components is very small.
Lorenzian functions, respectively. The broad component, as- The DQ filter can be set such to select the magnetization
sociated with the Gaussian line, corresponds to the rigid phase. only from the most rigid part of a heterogeneous sample. By
The Lorenzian line associated with the narrow component of choosing appropriate excitation/reconversion times τ (Figure 1)
the spectra describes the mobile phase. The intermediate line, of the double-quantum coherences, the magnetization corre-
described by the combination of Gaussian and Lorenzian sponding to the stronger dipolar couplings will pass through
functions, is associated with the interface. the filter and that of the weaker dipolar couplings is filtered
The phase composition for hard R-keratin, in the native state out. The optimum value of τ can be chosen by recording 1H
and after oxidative and reductive/oxidative treatments in D2O DQ build-up curves. The maxima of the DQ build-up curves
for the temperature range where denaturation takes place is appear at very short excitation/reconversion times τ of about
shown in Figure 5. The denaturation temperature of hard 10-12 µs for all investigated samples. In this range of τ values,
R-keratin is 154 °C, that for the hair samples after the oxidative the mobile component is completely filtered out, as shown
treatment is 122 °C, and that for the sample subjected to below.
Morphology and Molecular Mobility of Hard R-Keratins J. Phys. Chem. B, Vol. 113, No. 35, 2009 12141
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Publication Date (Web): August 6, 2009 | doi: 10.1021/jp904484r
2 lnπ 2 〈r 〉∆ν
value of the signal-to-noise ratio. 1
4.4. Proton Spin Diffusivities. An accurate analysis of the DR ) 2
1/2 (12)
12
domain thickness by NMR spin diffusion experiments requires
three steps. These are as follows: (i) an optimization of a dipolar
filter to obtain the highest selectivity to the different phases, and
12142 J. Phys. Chem. B, Vol. 113, No. 35, 2009 Baias et al.
1
DM ) 〈r2〉[R∆ν1/2]1/2 (13)
6
where R is the cutoff parameter of the Lorentzian line, ∆ν1/2 is
the full line width at half-height, and 〈r2〉 is the mean square
distance between the nearest spins. An estimation of 〈r2〉1/2 ≈
0.22 nm was given for keratin taking into account the amino
acid composition.13
The calculated spin diffusion coefficients using eqs 12 and
13 are shown in Figure 8. For each denaturation temperature
and type of sample, the specific values of DR and DM are used
for domain size evaluation. The largest value for DR showing
the highest organization and packing corresponds to hard
R-keratin (Figure 8a). The morphology dezorganization due to
reductive/oxidative treatment leads to a reduction of DR. This
trend is also valid for DM (Figure 8b).
4.5. Morphology and Domain Sizes. The spin diffusion
experiments were performed on native hard R-keratin and
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( )
DR + DM
〈z2〉1/2 ∝ 2 td (14)
2
Figure 12. Rigid domain sizes for rigid and mobile + interface
fractions of hard R-keratin with different treatments as a function of
denaturation temperature.
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Publication Date (Web): August 6, 2009 | doi: 10.1021/jp904484r
Figure 15. Enlarged version of the alkyl (a) and R-carbon (b) regions
of the 13C CPMAS spectra at a rotor frequency of 5 kHz for hard
R-keratin, in the native state and after oxidative and reductive/oxidative
treatments. The dashed line at 40 ppm marks the 13C resonance of the
cysteine engaged in the disulfide links. The 13C CPMAS spectrum of
the -S-S- bond free sample is also shown.
for the case of hard R-keratin with acetylated sulfur where the
line width of the carbonyl signal is slightly larger than the others,
suggesting that the acetylation of sulfur after breaking the
disulfide bond induced a certain degree of disorder in the hard
R-keratin. This supports our view of a three-phase model for
Publication Date (Web): August 6, 2009 | doi: 10.1021/jp904484r
Figure 17. Schematic representation of the intermediate filament (R-helices) imbedded in the keratin amorphous matrix (gray islands). The chemical
changes were induced by the oxidative (a) and reductive/oxidative (b) treatments (see text). RSH is the abbreviation for thioglycolic acid.
terminal domains projecting into the interfilamentous space and The chemical modifications we used for the keratin material
linking with the matrix proteins through disulfide bonds. The were focused on attacking the disulfide bonds. The oxidative
terminal domains contain, besides cystine, glycine, threonine, modification aims at breaking the S-S bonds and oxidizing
valine, alanine, and serine, acidic sites such as glutamic and them into cysteic acid (see Figure 17a). Under the reaction
aspartic acid. This scaffolding structure at the IF surface made conditions, not all of the bonds will be broken, but overall, it is
by the side-chain interactions that anchor the microfibrils to expected that both the scaffold and the matrix are crumbled.
the matrix (interface phase) assists the thermal stability and the As a result, the DSC peak corresponding to the denaturation of
primary control over the denaturation of the helical structure protein shifts toward lower temperature (around 130 °C, see
of keratin materials when heated. It has a protective role and Figure 3) and the enthalpy decreases compared to the original
the capacity to participate in the formation of a solid interface. keratin material. The interface amount for oxidative modification
The mechanism of thermal denaturation of keratins, as it has is reduced as compared to the hard R-keratin, as shown in Figure
been described ref 36 follows several steps. Beyond a certain 5a and b. Moreover, the molecular dynamics of side chains
temperature (the peak on DSC), the temperature rise leads to are intermediate between that of hard R-keratin and the sample
the breaking of the scaffold structure of IFs. At that temperature, subjected to the reductive and oxidative treatment (cf. Figure
the IFs are in a metastable state. The R-helix denaturates at 6). The rigid domain thickness is not essentially affected by
around 80 °C in soluble proteins, and it is only the interface the oxidative treatment (Figure 12).
that still keeps it organized. Once set free, the IFs (R-helices) The reductive/oxidative modification occurs in two steps.
denaturate. This involves a transition from a relatively compact First, the S-S bonds are broken by the action of the reductive
ordered structure to a more flexible, disorganized, opened reagent, thioglycolic acid (TGA), and then are reformed by the
polypeptide chain. As the process of denaturation proceeds, the oxidative reagent (see Figure 17b). During this sequence of
protein molecules unfold and the intern hydrophobic regions reactions, not all of the bonds are broken and not all of the
expose to the outside of the molecules. The hydrophobic groups broken bonds reform; besides, not all of the reformations occur
in water tend to cluster, leading to associations of molecules. at the same places. In other words, we expect to reform the
Morphology and Molecular Mobility of Hard R-Keratins J. Phys. Chem. B, Vol. 113, No. 35, 2009 12147
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