INFRARED MICROSPECTROSCOPY AND LINEAR DICHROISM
3.2 METHOD 1 Samples
Collagen fibres used in isothermal studies of dichroism were excised from a group o f untanned and semi-tanned hide samples that were also used for thermal analysis. Table 3.1 (see Chapter Four, section 4.2.1): (1) undeteriorated seal skin, (2) deteriorated seal skin, (3) undeteriorated brain-tanned deer skin, (4) undeteriorated brain- and smoke-tanned deer skin, and (5) photodegraded brain- and smoke-tanned deer skin
and (6) bovine Achilles tendon, BAT, (United States Biochemical Corp. Cat. 13815, Lot 53563) prepared by the method of Einbinder and Schubert (1951) and stored at +4°C. A segment o f seal skin thong (7) belonging to the Canadian Museum o f Civilization (nn708) was also analysed in this set. Microforceps and micro-surgical scissors were used. Care was taken to avoid applying tensile stress to the fibres and to avoid fraying them..
TABLE 3.1 Sample material including untanned and semi-tanned skins and vegetable- tanned leather.
Skin S am p les P rocessing A cronym
Seal (undeteriorated) Untanned, stretched before drying;
shaved, epidermis intact.
N ew Seal (N S )
Seal (deteriorated) Untanned, stretching unknown due
to deterioration
Deteriorated Seal(D S)
D eer (undeteriorated) Brain-tanned, sw ede-like Brain-Tanned D eer
(B T D )
D eer (undeteriorated) Brain tanning + sm oke tanning;
sw ede-like
Sm oke-Tanned D eer (S T D )
D eer (photodegraded surface layer) Brain tanning + sm oke tanning;
sw ede-like; photo-bleached
Photodegraded Sm oke-tanned D eer (P S D )
Seal Thong (photodegraded) Untanned, stretched before drying,
no fiir, no epidermis
Photodegraded Seal Thong (PS T )
B ovine A ch illes’ tendon Einbinder & Schubert (1951) B ovine A ch illes’
Tendon (B A T )
Leather Sconce Deteriorated, vegetable-tanned,
open grain
Deteriorated S con ce Leather (D S L )
Fire Fighters’ Breathing Apparatus Severely deteriorated vegetable-
tanned leather, closed grain
Deteriorated Apparatus Leather (D A L )
Dichroism was also studied over the shrinkage (dénaturation) temperature range. In addition to the samples listed above, fibre samples of leather were taken from— (1) an open grain vegetable-tanned leather wall-hanging (Prince Edward Island Museum and Heritage Foundation; Canadian Conservation Institute registration number (CCI No.)
1.000.795), and (2) a closed grain vegetable-tanned leather on a fire fighter's breathing apparatus which was the property of the Ontario Fire College Museum (CCI No.
2,005,329). Small samples of leather fibres were taken from two areas on the sconce. A third sample, approximately 2 x 4 x 1 mm, consisted of a full-thickness fragment o f the leather which had broken off of the piece covering a vertical wooden support. Three fibre samples were taken from the leather "hood" of the breathing apparatus and one from an inner collar. Samples of new, undeteriorated vegetable-tanned leather were analyzed for comparative purposes.
The single, full thickness fragment from the hanging had an initial pH of approximately 3 and, after soaking in excess distilled water overnight, a final pH above 4.0. The pH of the leather sample was therefore just within the range of intermediate pH values in which no significant reversible effect occurs on thermal stability and shrinkage temperatures. (Such a range of values also permits little hydrolytic activity in short term storage tests of high humidity.) The shrinkage temperatures were therefore taken under essentially standard experimental conditions which permits their comparison with values reported in the literature.
Vegetable-tanned leathers normally have pH values around 4.0. In experiments which use increases in temperature (40°C - 60°C) and high humidity, rates of
deterioration in vegetable-tanned leather increase dramatically when the pH of the leather drops below approximately 3.5. Notwithstanding, the association between pH and
deterioration may not always be clear. Reaction products of acid hydrolysis tend to raise the pH, and therefore deterioration may be extensive despite a comparatively normal pH, i.e. 3-4. Identification of the extractable, acidic components in the leather was not determined.
3.2.2 Infrared Microspectroscopy
A method of preparing the samples for polarized FTIR microspectroscopy was needed which would avoid altering collagen fibre morphology. High pressure to flatten the collagen fibres was avoided. Although flattening can improve some of the optical properties o f polymeric fibres, it can also induce changes in the crystalline composition of some fibre types resulting in changes in peak frequencies, intensities and shapes (Tungol et al 1990). In native collagen, pressure, along with sheer forces, can cause the individual fibres in bundles to twist and distort. This produces a more random
orientation of individual fibres within bundles, which, as is discussed below, affects the dichroic measurements if the randomness occurs beyond a certain limit of misalignment.
Separate studies were performed with fibre samples immersed during analysis in mineral oil and in distilled water. The former was used to produce full mid-IR spectra of the collagen fibres but also, in a preliminary way, to determine how a perturbing agent of molecular structure, such as water, affects the dichroism of the undeteriorated and
structure resulting from deterioration (Marcott et a l\9 9 \, Mantsch et al 1986) and possibly remove potential ageing (annealing) effects (Bresse 1986, Struik 1978). Mineral oil does not perturb collagen and therefore provides an appropriate control for
determining the enhancing effects of the water. The oil-immersed fibres contained a moisture content at equilibrium to laboratory conditions; excessive drying was avoided to avert the slight changes to dichroism which have been reported (Bradbury et al 1958, Doyle et al 1975). Both media further served to reduce the amount o f radiation scattering at the sample during the collection of spectra.
3.2.2.1 Analytical procedure 3.2.2.1.1 Isothermal studies
Infrared analyses of the collagen fibres were undertaken using polarized and unpolarized radiation, both at ambient experimental temperatures and elevated
temperatures. Analysis by unpolarized radiation utilized low pressure diamond anvils in order to flatten acetone washed fibres sufficiently to produce a satisfactory pathlength for transmission studies. Polarization analyses were done in two ways. One utilized a single barium fluoride window to support fibres sitting in a film of spectroscopic grade mineral oil. Most of the studies, however, involved fibres washed in distilled water and placed between two barium fluoride (Bap2) (n=l .46) windows in additional water. A plastic
film gasket of plasticized poly(vinyl chloride)was placed between the two windows and around the fibres at the periphery. This sample assembly was then placed into a
to press the windows against the larger fibres and to seal the plastic gasket to prevent the evaporation of the water during analysis.
3.2.2.1.2 Thermal studies
Some of the investigations required heating the fibre samples through the dénaturation temperature range. The Spectra-Tech window support (Heated micro sample press, with 0019-025 temperature controller, Spectra-Tech, Inc.) with fibres incorporated as described above was heated to predetermined temperatures (+/- 1°C) at which spectral collection took place. In this fashion, fibres were heated in small steps generally through the range of 23°C to 80°C to collect spectra. From these, dichroic ratios were derived and plotted as a function of temperature to produce dichroic profiles of fibre dénaturation.
3.2.2.2 Collection of spectra
Fibres on window supports were placed onto a rotatable stage of a Spectra-Tech Inc., IR-Plan Research Microscope accessory, equipped with a high sensitivity, narrow band MCT (mercury-cadmium-telluride) detector. The microscope was coupled to a Bomem MB 120 infrared spectrometer. The IR radiation was polarized using a zinc selenide wire grid, positioned between the globar (silicon carbide) infrared source and the collagen fibre sample. Spectra were collected at a resolution o f 4 cm'', with 100 to 400 coadded scans, using a 15x reflecting Schwarzchild-type Cassegrainian condenser lens and a similar lOx objective lens. The effective numerical aperture of the microscope
optics was comparatively low at 0.58.
The visible optical path of the microscope was parfocal and colinear with the infrared path (Messerschmidt 1987). Visible-light polarizing optics were used to examine and align the fibre samples for analysis. The visible light analyzer (a polarizer placed above the sample in the optical path of a polarizing microscope) was rotated into a position parallel with the wire grid infrared polarizer. A portion of a fibre to be analyzed was first centered in the field of the microscope (Levy 1988). The visible light polarizer (the counterpart o f the polarizer pair used in a polarizing microscope, this one placed below the sample in the optical path) was then rotated to a crossed position to produce extinction in the visible field. The axial direction of the fibre portion was then positioned in parallel alignment with the wire grid infrared polarizer by rotating it to a position where its optical birefringence showed the maximum possible extinction in parallel with the visible light analyzer (Figure 3.1). The fibre portion was then isolated for IR
transmission microspectroscopy by the use of two adjustable remote apertures (Messerschmidt 1987). With these properly adjusted, the fibre was carefully moved aside to record the parallel and perpendicular reference spectra from the medium (water) just beside the fibre. The fibre portion was then moved back into center position to
accumulate the two raw sample spectra. During the analysis, the fibre sample was left stationary. The window created by the remote apertures remained the same both in size and orientation, and the wire grid polarizer was rotated to produce the parallel and perpendicular spectra. Two transmittance spectra, one for each orientation of the
polarizer, were produced by taking the ratio of each raw sample spectrum with the corresponding
reference spectrum. The transmittance spectra were converted to absorbance spectra. The perpendicular absorbance spectrum was subtracted from the
\ \
Figure 3.1 Deteriorated collagen fibre show ing max. possible birefringence (left) and extinction (right) under crossed polarizers. Rectangle marks typical area for analysis; parallel spectrum (right) and perpendicular spectrum (right). Reference spectra were taken for both orientations just to the side o f the fibre.
parallel spectrum to produce dichroic spectra. "Spectra Calc" (Galactic Industries Corporation, Salem, New Hampshire) software was used for two-point baseline corrections and to integrate the amide III band (1307 - 1135cm ') for the parallel (A,) and perpendicular (A^) orientations. The values of the integrations were used to produce a dichroic ratio {A JA J for the analysed fibre.
3.2.3 Microscopic Shrinkage Temperature Measurements
Samples of collagen fibres were taken from the various skin and leather materials and tested by the method described in chapter two and previously published, Young (1990). Fibres were soaked briefly in acetone and then in distilled water for about ten minutes, then heated in excess water at a rate of 3°C/min in a microscopical hot stage. The temperature range over which shrinkage occurred was recorded.
3.3 RESULTS
In presenting the experimental results, reference is frequently made to other work in order to show how the current findings fit with present knowledge. The results are presented in two parts. The first introduces the major mid-infrared absorbance bands of collagen fibres and gives special attention to the amide III band complex. The
discussions include evidence from isothermal (ambient) experiments and heating and dénaturation experiments in characterizing the sensitivity of unpolarized amide III absorbance to changes in collagen molecular conformation.
The second part continues with a more in-depth examination of amide III using polarized radiation in comparisons of deteriorated and undeteriorated fibrous collagen in terms of conformational change in response to heating and dénaturation.
3.3.1 IR Absorbance Spectra of Collagen Fibres
Mid-IR spectra o f diamond-anvil-sampled collagen fibres are illustrated in Figure 3.2. The major absorption bands result from the vibrational modes of the amide functional groups along the peptide chains of the coiled-coil triple helical molecule of collagen (Morton 1975, Fraser and MacRae 1973a,b) Table 3.2 lists the bands and their from the undeteriorated seal skin, seal skin thong and deteriorated seal skin. Subtle differences occur in the shape, frequency and intensity of many o f the absorbance bands. Some may be the result of molecular change due to the pressures used to flatten functional group assignments. The three spectra of Figure 3.2 represent fibres taken the fibres in the diamond-anvil sampling technique. When sample preparation does not
N e w s e a l (N S ) 0 Ü c (T3 O (/) < S e a l t h o n g ( P S T ) D e g r a d e d s e a l (O S ) 3000 4000 3500 2500 2000 1500 1000 W a v e n u m b e r ( c m ' ^ )
F igure 3.2 IR spectra o f undeteriorated (NS), photo-deteriorated (PS T ) and deteriorated skin (DS).
alter structure and therefore does not confound the spectral data, many of the spectral differences are of value in detecting chemical and conformational change. For example, broadening of the amide A band at 3320cm'' (attributed to N-H stretching vibrations), so that the band includes a greater component at 3300, indicates dénaturation (Fraser and MacRae 1973a) or possibly deterioration in the system. In the absence of deterioration, the effect likely results from the rearrangement of hydrogen bonding from a regular inter-chain pattern to an irregular pattern, including increased hydrogen bonding to
neighboring water molecules (Bower and Maddams 1989), and the breakdown in the resonance coupling of amide A vibrations among the peptide groups along the peptide
Table 3.2
IR Absorbance Peaks for Solid Phase Collagen
FREQUENCY (cm ') IDENTIFICATION 3450-3400 OH in free water 3290-3330 N-H Stretch 3060-3100 Overtone of Amide II 2930-2950 C-H Stretch 2870 C-H Stretch 1710 Non-ionized COOH 1640-1660 C=0 Stretch (80%) (Amide I) 1560 COO
1535-1550 N-H in-plane deformation and C-N
stretch (Amide II)
1445-1455 CHj deformation and CHj
asymmetric deformation
1407 COO
1375-1391 CHj symmetric deformation
1310-1340 CHz wagging
1230-1270 C-N stretch (40%) and N-H in
plane deformation (30%) (Amide III)
1075-1082 CO vibrations o f hydroxyl groups
940 0-H deformation of COOH
920 0-H deformation of COOH
chain. Because collagen fibres also contain some amount of glycoprotein and lipid, some o f the distinguishing features in the spectra are likely due to differences in the quantities or chemical state of these non-collagenous components.
Amide I at approximately 1650 cm ' is also sensitive to conformational change. Fourier transform spectral self-deconvolution reveals three absorbance peaks, at 1633,
1643 and 1660 cm ' in amide I for both collagen and gelatin (denatured collagen) in solution (Payne and Veis 1988). Though the peaks persist through dénaturation, their relative intensities change with the extent of triple helix content. The peak at 1660 cm ' dominates the spectrum of native collagen, but with dénaturation its intensity drops and the peak at 1633 cm ' increases and dominates.
The amide III band complex of collagen fibres contains prominent overlapping peaks at 1284,1242 and 1205 cm '. Two point baseline correction at 1308 and 1135 cm ', along with fourier transform self-deconvolution using a value o f 1.23183 for the
exponential filter, gamma, and 0.72890 for the smoothing filter (bessel apodization) produced the amide III complex shown in the middle of Figure 3.3. Further processing of this spectrum was undertaken to produce 2nd and 4th order derivatives (Koenig 1992, Maddams and Mean 1982), incorporating an essential, minimum of Savitsky-Golay smoothing, and these are shown above and below, respectively. Peak amplitude in the derivative spectra is a function of the original peak width with a bias to sharper original peaks. Derivatization produces lobes of opposite sign on each side of the real peaks, and therefore adjacent real peaks located at lobe frequencies can be partly or completely lost (Koenig 1992). In the derivative spectra, the central, dominant peak at 1242cm ' consists
s c CO -e o </) W a; co & 0) > ro > <D TJ •O C CM I V A I (0 & 0) > ro > _ L _ l 1350 1325 1300 1275 1250 1225 1200 1175 1150 W a v e n u m b e r (cm
F igure 3.3 Two-point baseline corrected, self-deconvolved amide III band (middle); 2nd derivative o f band (top) with peaks pointing down; 4th derivative (bottom) with peaks pointing up.
of three overlapping peaks, the approximate locations of which are listed in Table 3.3. Amide III is particularly sensitive to conformation in fibrous proteins (Fraser and MacCrae 1973a), showing characteristic frequencies for the various structural
organizations of peptides including— alpha helices, beta conformations, and collagen's triple helix. In collagen, amide III is sensitive to changes in the state of aggregation and,
Table 3.3
Peak Maximum Amide III Frequencies Identified by Derivative Spectroscopy N a tiv e , undeteriorated co lla g en fibre N a tiv e, deteriorated co lla g en fibres D enatured, undeteriorated co lla g en fibre D enatured, deteriorated c o lla g e n fibre 1 2 05.4 1205.4 1202 1 2 0 3 .4 1 2 3 2 .4 1 2 32.4 12 3 0 .4 1240.1 1239 1 2 43.9 1243 1255.5 1255.5 1255.5 1 2 5 3 .4 1267.1 1267.1 1267.1 1 2 6 5 .2 1 2 84.4 1 284.4 1284.4 1 2 8 4 .4 130 3 .7 1303.7 1305.7 1 3 0 1 .8 1323 1319.2 1323 1 3 1 3 .4
(Self-d econvolved and corresponding derivative spectra used to obtain the peak positions listed in Table 2 are presented in A p p en d ix C.)
as will be shown later, to molecular conformational change resulting from phase transition. Jakobsen et al (1983), for example, have shown changes in overall intensity of the amide III complex and in the ratio between peaks at 1257 cm ' and 1242 cm ' in undeconvolved spectra. In solution, at 4°C, with no intermolecular interactions, native collagen molecules show a 1242/1257 cm ' ratio of less than one. At 30°C, fibrils form spontaneously, the absorbance over the entire amide III band increases markedly, the ratio invert and is so high that the reported 1257 band exists only as a shoulder on the 1242 band. As is revealed below, the band complex is also sensitive to deterioration-
induced conformational change. The results that follow are presented from the viewpoint o f losses in order and structure as opposed to their development as with the work of Jakobsen et al (1983).
3.3.2 Changes in Band Shape and Intensity
Baseline-corrected amide III band regions of deteriorated and undeteriorated seal skin collagen fibres in water are depicted in Figure 3.4, both before and after
hydrothermal dénaturation. Spectra were choosen in which the amide III bands showed absorbance intensities of less than approximately 0.8. All measurements were taken at ambient temperatures. The peak height absorbance of the deteriorated, undenatured sample was adjusted to match that of the undeteriorated, undenatured sample at 1242cm'* using a suitable scaling factor, k, to satisfy the equation-
Au - kAj = 0 = - kfldQ^d (4.3)
where A„ is the peak height absorbance of the undeteriorated sample and Aj the absorbance of the deteriorated sample, ‘a ' is the absorptivity coefficient; ‘C ’ is the concentration o f the absorbance band; and 'b ' is the thickness of the sample. By the nature o f the sample preparation, thickness was approximately the same for the two samples. Changes both in absorptivity and concentration likely account for the lower absorbance in deteriorated samples. The cause is likely peptide scission and greater structural disorder, both of which reduce resonance coupling and the number of
undenatured, undeteriorated denatured, undeteriorated undenatured, deteriorated denatured, deteriorated / 0.8 0.6 8 c TO
I
0.4 W < 0.2 0.0 1350 1300 1250 1200 1150 W a v e n u m b e r (cm ‘ ^)Figure 3,4 Unpolarized IR o f A m idelll band intensity with dénaturation: 1. undeteriorated before (solid) and after (dash), 2. deteriorated before (dot-dash) and after (dot-dot-dash).
secondary amides, the only amides which contribute to the amide III absorbance band. Low dichroic ratios for such fibres, reported in section 4.3.6, confirm the structural breakdown. The spectrum taken of the deteriorated sample after heat dénaturation was