INTRODUCTION
The cell walls of higher plants are structurally complicated, mostly polysaccharide, matrices which are economically important because they modulate cell structure, morphology and act as a barrier to small molecules (Darvill et al. 1980). Because of these characteristics, methods which proffer the ability to observe perturbations in the microscopic structure of the wall matrix, as a function of various treatments, could impact the processing and storageability of plant products. The primary cell wall of most higher plants is a biphasic framework consisting of a infrastructure of cellulose microfibrils held together by a rigid gel-like lattice of matrix polysaccharides (Preston 1979). These matrix polymers, which make up approximately two-thirds of the total primary cell wall and middle lamellar mass, are hydrophilic, polyhydroxy macromolecules which are extensively hydrated in vivo. Galacturonic acid-containing matrix polysaccharides in apples (Barrett & Northcote 1965, Ben-Arie et al. 1979, Stevens & Selvendran 1984), frequently referred to as pectin or pectinic acid, are based on linear blocks, ca. 40-80 monomer units long (Irwin et al. 1988), of [[alpha]]-(1->4)-linked D-galacturonic acid (homopolygalacturonans) interspersed with (1->2)- and (1->2 or 4)-substituted L-rhamnose. The L-rhamnoses are usually further linked with neutral polymers forming side-chains. Further complicating the molecular picture is the fact that the C6 carboxyl groups of the polyuronides are methyl esterified 50-70% (Irwin et al. 1985a) and possibly reside block-wise (Jarvis 1984, Irwin et al. 1988) in hydrophobic domains.
One of the more important functions of pectin in situ relates to its capacity to act as a cation exchange resin, thereby modulating mono- or divalent cation activity within the cell wall network. These acidic polysaccharides also manifest a cooperative-sequential binding mechanism (Irwin et al. 1984, Irwin et al. 1985b) for divalent cations. The most characteristic physical property of the pectinic polysaccharides is their ability to form gels and aggregates under aqueous conditions (Grant et al.1973, Cesaro et al. 1982, Fishman et al. 1984). The mutual interactions and attractions of various polysaccharides have been investigated in gels (Dea et al. 1977) and different types of intermolecular associations can be distinguished. Certain gel structures have been proposed to prevail through the exclusion of portions of the polymer chain and the realignment of stiff structures with more compatible geometries which result in mixed aggregates. The gel structure of polymer chains in cell wall matrices is much more difficult to ascertain because of the multifarious interaction of weak forces between adjacent chains. Thus, the higher order structure of acidic polysaccharides in their natural, hydrated-solid, state are not well understood because hydrogen bonding, hydration effects and dipolar/ionic forces may interact in manifold ways to secure these macromolecules within the matrix in ordered arrays (Rees et al. 1982).
Electron paramagnetic resonance (EPR) spectroscopy is a premier technique to characterize the microscopic and dynamic features (Berliner 1976) of numerous chemical species. Nitroxyl spin probes have been used widely to procure data about localized molecular attributes such as conformation, flexibility, polymer-diluent interactions in diverse systems. Only within the last several years has a significant amount of research on spin labeled carbohydrates been published (Gnewuch & Sosnovsky 1986). Even less information is available about the utility of these methods for obtaining information on the microrheological properties of foods as will be discussed in this chapter. To this end, we have opted for the covalently-bonded spin label method to study the rotational freedom between adjacent pectin molecules in apple cell walls (CW) or polygalacturonic acid (PGA) chains as they approach equilibrium hydration as a function of various levels of bound Ca[2+] and/or degree of methyl esterification (Irwin et al. 1987, Chamulitrat et al. 1988, Chamulitrat & Irwin 1989, Irwin et al. 1991).
BASIC PRINCIPLES OF NITROXYL AMIDE SPIN LABELING
Excellent books (Swartz et al. 1972, Poole 1983, Wertz & Bolton 1986) have been devoted to the description of the basic EPR experiment and all its associated hardware; it is pointless to belabor what these fine texts do so well and to these the reader is referred. Most spin labels are nitroxide free radicals which have the general structural formula
R1--C(CH3)2--[NO]*--C(CH3)2--R2 .
In this formula, R1 and R2 are side groups which give the nitroxide radical a certain degree of specialization for particular reactions with various functional groups (Wertz & Bolton 1986). In our case, R1 and R2 are connected together to form a heterocyclic ring with a free amine, 4-amino-TEMPO or 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4AT), which we use to form an amide linkage with the C6 carboxyl group (Figure 1 insert; Irwin et al. 1991) of D-galacturonic acid in PGA or CW pectin. The methyl groups, which surround the N--ODBA17()[*] moiety, bestow a certain amount of protection of this functionality from being reduced rapidly to its hydroxyl amine (N-OH) form. Figure 1 shows a semi-log plot of spin concentration of this same sample over a three year period when stored dry at room temperature; the bottom inset figure shows the calibration, necessary to perform periodically, to determine the best location within the microwave cavity for quantitation. These results reveal that our nitroxyl amide spin label, compared to other organic free radicals, is relatively stable over a long period of time ([[tau]]1/2~2 yrs. under less than ideal conditions). However, in the presence of a strong reducing agent, such as ascorbic acid (Figure 2; Irwin et al. 1991), this same compound is reduced ([[Delta]]G[300K] = -4.12 kcal/mole) to its hydroxyl amine form within a few hours.
The type of EPR spectrum obtained for a nitroxide label depends on the chemical environment surrounding the label (e.g., hydrophobic, hydrophilic) as well as the rate ([1]/[[tau]]R in units of s[-1]) at which the nitroxide group can reorient or rotate about its magnetic x axis (Figure 1 insert). If the nitroxide group is completely free to rapidly reorient about this axis ([[tau]]R ca. 10[-9] s or less) one observes a simple triplet (for our PGA nitroxyl amide label in solution Aisotropic = F(1,3) Tr A DBa10()[~] ~ 16 G and [[tau]]R ~ 0.9-2.8 ns; Irwin et al. 1987), due to the electron-nuclear hyperfine coupling with quadrupolar N (I = 1). As the environment surrounding a spin label changes, or becomes more rotationally restrictive, the line shape of the spectrum changes because molecular motion does not sufficiently average anisotropies in the local environment, resulting in broadened line widths and non-zero off-diagonal matrix elements for g DBa10()[~] and A DBa10()[~] (tensors of the 0[th] [scalar], 1[st] [vector] and 2[nd] ranks are herein represented as n, n or n DBa10()[~], respectively).
The Covalent Attachment of the Spin Label, 4AT, to Acid Sugars
The general reaction (Hoare & Koshland 1967, Taylor & Conrad 1972) of water soluble carbodiimides with polyuronides and subsequent nucleophilic attack by 4AT is shown in Figure 3. In all the labeling performed herein (Irwin et al. 1987, Chamulitrat et al. 1988, Chamulitrat & Irwin 1989, Irwin et al. 1991) the carbodiimide utilized was 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Previously, Hoare and Koshland (1967) have shown that reaction 1 (Figure 3) proceeds rapidly with the uptake of ca. one mole of H[+] per mole of acid depending on the ionization constant ( F([A-],[AH]) ) and the reaction pH (Taylor & Conrad 1972; Figure 4); this latter notion is readily supported by the fact that the predicted ionization constants (Daniels & Alberty 1974) were close to those derived experimentally. In the presence of a high concentration of a strong nucleophile, such as 4AT, reaction 2 proceeds with the formation of our nitroxyl amide. High resolution [13]C NMR (Irwin et al. 1987) spectroscopic data of the reaction products show no evidence of N-acylurea (Figure 3, reaction 4).
The Spatial Organization of Nitroxyl Amides in Acid Sugar Polymers
Interestingly (Irwin et al. 1987), upon reaction and subsequent dialysis, we observed a significant degree of line broadening in partially reacted PGA powders (Figures 5 and 6) arguing that the 4ATs were selectively reacting with carbodiimide-activated carboxyl groups in relatively small blocks or linear arrays rather than randomly throughout the polyanionic matrix. Generally speaking, when a system of electron spins, S, interacts with an applied magnetic field, H (Happlied + Hlocal), to produce a resonance line, each magnetic moment precesses about the z component of H with a frequency proportional to Hz. The scalar value as well as orientation of H will vary from one label to the next if there is a high spin density, which results in a significant Hz,local field contribution, and causes a smearing out of the observed precessional frequencies thereby resulting in line broadening (Pryce & Stevens 1950). Such dipolar broadening is theoretically described by the Hamiltonian (H ) operator,
Hdipolar = g [2][[beta]] [2] ISU(i>j, , )BBC{(F(Si*Sj,rS(3,ij)) - 3 F([rij *Si][rij *Sj],rS(5,ij))).
In this relationship g, [[beta]] and S have their usual meaning (Van Vleck 1948, McMillan 1968, Abragam & Bleany 1970, Leigh 1970) and r is the distance vector between the i[th] and j[th] spins. The summation subscript, i>j, identifies that each pair of dipolar interactions should be counted only once. Exact computation of Hdipolar is not feasible but can be statistically evaluated by the method of moments first developed by Van Vleck (1948). The second moment ([H[2]>ave), which is related to a Gaussian's first derivative line width ([[Delta]]Hpp = 2[H[2]>aveDBA16()[1/2] ), is defined as
[H[2]>ave = I(-*,*, (H - Ho)[2] f(H) dH) /I(-*,*,f(H) dH).
Figure 5 shows digitally calculated [H[2]>aves for typically broadened 1[st] derivative nitroxyl amide PGA (Figure 5, insert spectrum) powders and corresponding rigid limit simulations as a function of an empirical broadening parameter, F(d1,d). If one considers calculations for a randomly-oriented lattice (Van Vleck 1948, Irwin et al. 1984, Irwin et al. 1985b) the following relationships are true:
[H[2]>ave = F( 3, 5) g[4 ][[beta]][4 ]h[-2] S(S + 1)ISU(i>j, , )F(1,rij[6])
and
ISU(i>j, , )F(1,rij[6]) = BBC{(F([[kappa]][2],d[6])).
Thus, assuming that only dipolar spin-spin interactions occur, [H[2]>ave is related to the reciprocal 6[th] power of the distance between a group of i- j[th] interacting spins. In the above equations d is the nearest neighbor distance parameter for an ordered array and [[kappa]] varies with the lattice disposition. We have utilized the [[kappa]] term because changes in [H[2]>ave cannot be attributed entirely to the nearest neighbor distance parameter. As a paramagnetic species begins to fill the lattice, an increase in the number of dipolar spin-spin interactions will occur at each i[th] spin. For example, [[kappa]] = 1 for two interacting spins, 1.4 for a linear array, 2.45 for a two-dimensional hexagonal array with six nearest neighbors and [[kappa]] = 3.46 for a three-dimensional hexagonal close packing array with 12 nearest neighbors. Thus, considering nearest neighbor interactions only, [[kappa]][2] is an estimate of the number of dipolar interactions at distance d even in a system with an extended array due to the decreased weighting of distant interactions. As [[kappa]] approaches 1, we have found that [[kappa]][2] provides an accurate estimation of the number of near neighbor interactions per nitroxyl amide (Figure 6; Irwin et al. 1988) since, in this low concentration region, the number of distant interactions would be negligible. We can approximate the dimer-only nearest neighbor distance parameter from the extrapolated zero concentration-[H[2]>ave intercept (Figure 6 arrow); at this value of [H[2]>ave, [[kappa]] is about one. Assuming that d remains relatively constant with addition of the paramagnetic species we can calculate the number of strongly interacting spins per i[th] point dipole using the expressions above from each of the empirically-derived values of [H[2]>ave. The concentration of bound paramagnetic species at which the number of dipolar spin-spin interactions is approximately one is related to the size of the homopolygalacturonan blocks since the probability of bonding at near neighbor sites is far more likely than any other position within that same block. Because of this type of amide bonding we can assume, as a first approximation, that one spin label pair bonds per polymer block within the matrix when [[kappa]] is approximately 1. If this assumption is true the polymer's average degree of polymerization (O([--],DP)) can be estimated from SUP3([[chi]]) when [[kappa]] = 1,
BBC{([1]/[[chi]] )[[kappa]]~1 = O([--],DP) ,
whereupon SUP3([[chi]]) is the mole fraction of paramagnetic dimers bound. For PGA the O([--],DP) was found (Irwin et al. 1988) to be ca. 36 and approximately agrees with the O([--],DP) obtained from reducing end group titration (Fishman et al. 1984) for these same compounds. The electron spin-lattice relaxation time (T1) is related to interspin distances (Eaton & Eaton 1978, Hyde & Rao 1978). If the nitroxyl amide line broadening discussed above was due to relatively short interspin distances, as we maintain, the disruption of the PGA nitroxyl amide lattice, through ascorbate reduction (Figure 2) or by competitive reactions with a nonparamagnetic amine of similar size (e.g., aniline), should induce a corresponding increase in a parameter, R(T1T2)/T2,est (= T1*; 1/T2,est = {[[gamma]] R(3) [[Delta]]Hpp}/2), related to T1 ; T1* is calculated from changes in the relative intensity of the centermost 1[st] derivative N--ODBA17()[*] component of the low power portion of a power saturation experiment, as described by Poole (1983, p. 593; see Figure 11 for a typical saturation experiment). The data in Figure 7 clearly illustrate this principle. Upon reducing part of the nitroxyl's spin or reacting PGA with both 4AT and aniline, in the presence of EDC, the effect on relative T1* was substantial. The nitroxyl amide T1*s increased by a factor of about 4 upon reduction with ascorbate to a total spin concentration of approximately 0.6 mole % N--ODBA17()[*]
([N--ODBA17()[*] ]/[N- OH+N--ODBA17()[*] ] =0.055). The relaxation time parameter was even more influenced in the aniline reacted polymer than by partial reduction; this observation is ostensibly due to the fact that aniline has a smaller pKa than 4AT and therefore more amines are available to react (e.g., unprotonated) at this pH (4.75) therefore more anilide functional groups are covalently bound than reduced 4AT-amides and result in greater nitroxyl amide spacing. It is also likely that the reduction of the nitroxyl amide polymer is not completely random since the slope of the line, shown in Figure 2, is less than unity (e.g., negatively cooperative, Irwin et al. 1991).
RESOLVING PGA AND CW NITROXYL AMIDE "LOCAL" AND "INTERNAL" MOTIONS
Dehydrated PGA's nitroxyl amide spectra, measured at 77 K, were simulated using a rigid limit program (Chamulitrat et al. 1988) to obtain accurate g DBa10()[~] and A DBa10()[~] tensor components. These simulations applied Simpson's numerical integration over [[theta]] and [[phi]] which are the angles between H and z themselves as well as their projection in the rotating frame. The spectral parameters, Azz and gzz, were measured empirically from the separation between the outer hyperfine extrema (2Azz) and the mid-point of the two extrema, respectively. The orientationally dependent line width used to fit spectra had the form of
F(1,T2) = [[alpha]] + [[beta]]cos[2 ][[theta]].
A Lorentzian line shape provided the best simulations of the experimental EPR spectra. Parameters which gave the best fit were: Axx =7.0 G, Ayy = 4.5 G, Azz = 35.8 G; gxx = 2.0095, gyy = 2.0059, gzz = 2.0022; the orientation dependent line width parameters, [[alpha]] and [[beta]], were found to be 5.8 G and 0.2.
Detailed simulations (Chamulitrat et al. 1988), using Stochastic Liouville theory (Freed 1976), indicated that the preferred axis of rotation, x, was about the magnetic y axis of the nitroxyl amide (Figure 1, insert). These simulations were performed assuming that the rotational diffusion tensor, R DBa11()[~] , was axially symmetric about x. The best simulations of this type show that the nitroxyl amide moiety's preferred motion, in the molecular frame, was approximately about PGA's main axis. The best model for reorientation was the moderate jump. At low temperatures spectral line shape was mainly sensitive to local motions predominantly about the NH-C4 bond since the CO-NH bond had an appreciable double bond character. An energy of activation (Ea) of 0.39 kcal/mole (Figure 8, open circles; loge{1/[[tau]]R} = loge{1/[[tau]]R,o} - Ea/RT) was estimated for the temperature range 141 to 271K from the detailed simulation-derived [[tau]]Rs. For comparison purposes, empirical [[tau]]Rs (Figure 8, open squares) were also calculated as
[[tau]]R = a BBC{(1 - F(Azz'i,Azz'rl))SUP6( b)
whereupon Azz'i is Azz observed at a given temperature and Azz'rl is that of the rigid limit; the parameters a and b are supplied in Table 1 for different models of diffusion and line widths. Henceforth, for simplicity sake, the low temperature range [[tau]]Rs, where Ea is small, will be alluded to as [[tau]]Rs due to "local" motions while those in the higher temperature range will be referred to as "internal" motions since the latter type are predominantly due to reorientation about the axis which closely corresponds to PGA's main chain. Structural evidence that the higher temperature range [[tau]]Rs were due to the nitroxyl amide's reorientation along the homopolygalacturonan main chain is presented in Figure 9 (Chamulitrat & Irwin 1989) where there was observed to be an increase in Ea as a function of Ca[+2]DBa9()bound in both PGA and native apple cell wall matrices. These data indicate that Ca[+2] cross links more efficiently in PGA than in the CW matrix probably because the native polyanionic lattice is disrupted by methyl esterified uronosyl monomers as well as relatively large side chains. Enzymatic deesterification (Table 2) of the apple CW caused the internal motion Eas to approach those of PGA and argues that the nitroxyl spin labels are providing spatially specific information on Ca[+2]s effects at or near cell wall methyl ester domains. When our nitroxyl amine is specifically reacted (Irwin et al. 1988, Chamulitrat & Irwin 1989) with the cell wall matrix acid sugar polymers (Figure 10, upper-most spectrum) to form the nitroxyl amide the Cross Polarization and Magic Angle Sample Spinning NMR (CPMAS/NMR) methyl ester resonance disappears; this apparent broadening effect is completely reversible by reduction of the nitroxyl (N--ODBA17()[*] ) to the hydroxyl amine (N- OH) with ascorbate (Figure 2, bottom spectrum). The illusory diminution of this methyl ester resonance could be due to several mechanisms (Redfield 1965, Eaton & Phillips 1965, Gerasimowicz et al. 1984): the paramagnetic amide's effect on [1.] rotating-frame spin-lattice relaxation, [2.] the spectral density of this resonance or [3.] "T1-driven T2*" whereupon that part of the free induction decay (FID) associated with the O- methyl resonance was not sampled due to a rapid loss of the FID envelope in the "dead time" prior to data acquisition. Regardless of the mechanism, these data support the hypothesis that the nitroxyl amides react close to the methyl ester hydrophobic domains. Interestingly, CW nitroxyl amide's relaxation properties were not much modulated by Ca[+2] cross links (Figure 11; Irwin et al. 1991) since our relaxation parameter, R(T1T2), as measured by power saturation, were similar in Ca[+2]-treated and control matrices. However, for inhomogeneously (anisotropic interactions in randomly oriented systems in the solid state) broadened samples such as these, Metz and coworkers (1990) have recently demonstrated that perhaps a better measure of spin-lattice relaxation (R(T1T2*)) can be obtained from the microwave power where maximal signal intensity (PuDBa5()[max]) is observed, assuming: [1.] a Lorentzian line shape, [2.] H1 (the microwave field scalar value in units of G) = R(Pu) (in units of watts, Poole 1983)
BBC{(T1T2*)SUP6(F(1,2)) [[proportional]] BBC{(1/PuDBa6()[max])SUP6(F(1,2)) ,
and [3.] T2* (a measure of H inhomogeneity) remains the same for similar samples, such as ours, and is much less than T2. For the Ca[+2]-treated PGA (Figure 11 insert: squares) sample the PuDBa5()[max] was ca. 22.18 mW while that of the control (Figure 11 insert: diamonds) was ca. 34.68 mW indicating that the relative R(T1T2*) s, as defined above, were approximately 20% larger after the Ca[2+]-treatment. Accurate PuDBa6()[max]s were obtained from the value of R(Pu) where the 1[st] derivative empirical biexponential curve fit ({dI(H)/dH}max-vs-R(Pu) ) with respect to R(Pu) approached 0. Relaxation parameters can also be calculated (Figure 12; Irwin et al. 1991) from the nonlinear numerical method of Metz and coworkers (1990); this method is problematic, especially with relation to the spin-spin relaxation time, T2, as exemplified (Figure 12 inset) by the fact that T1 and T2 are positively linear,
slope = T2*DBa8()[2] BBc{(F(dT1,dT2)) = +0.34 (assuming T2* constant),
with respect to each other. Of course, one would expect that as the uronosyl nitroxyl amide [[tau]]Rs for internal motions increase (e.g., [[tau]]R,CW > [[tau]]R,CaPGA > [[tau]]R,PGA) to the point where T1 > T2, T2 should subside (Bloembergen et al. 1945) as T1 and [[tau]]R increase. Regardless, this new technique does provide a reasonable way to measure R(T1T2) s without assuming H1 is equivalent to R(Pu) which was required by the linear technique displayed in Figure 11.
EQUILIBRIUM HYDRATION-INDUCED CHANGES IN MAIN CHAIN MOTION
Under ordinary circumstances, the primary wall and middle lamellar network of apple cortical cells are moderately- (30-50% [w/w]) to fully-hydrated ( >50% [w/w]; Chamulitrat et al. 1988). The higher order structure of cell wall matrix homopolygalacturonans are modulated to a significant degree by bound water molecules (Irwin et al. 1985b). Knowledge about the spatial deformations, as measured by relative molecular flexibilities at different levels of hydration, may assist in the clarification of the higher order structure of sugar acid containing matrix polysaccharides.
Both CW and PGA nitroxyl amides hydrate over time at 100% relative humidity in a remarkably similar fashion (Figure 13; Irwin et al. 1991) considering their extreme disparity in molecular constituency (e.g., only ca. 25% of the CW is made up of uronosyl residues and a large portion of these are methyl esterified; Irwin et al. 1985a) albeit the equilibrium level of hydration for PGA was ca. 25% greater. In order to analyze the hydration process from the standpoint of the nitroxyl amide moiety (Figure 14), we have determined both the number and electron-nuclear distance (r) of nitroxyl amide bound [2]H2O in hydrated spin labeled PGA. These data were obtained at 4.2K using a three-pulse electron spin-echo modulation (ESE) pulse sequence (Kevan & Schwartz 1979) on moderately- (top set; 30% [w/w] [2]H2O) and fully-hydrated (bottom set; >=50% [w/w] [2]H2O) nitroxyl amides. Simulations of the ESE modulation envelopes indicate that up to 4-8 [2]H2Os bind equivalently to each nitroxyl moiety at approximately 3.6 Ĺ with an Aisotropic approaching 0 MHz. The computer simulation of these ESE envelopes was not facile as there could have been spectral distortion of the initial part of the time domain array due to a small inner layer of bound [2]H2O with a nominal r and nonzero Aisotropic . This potential problem is made more clear upon Fourier transformation of the time domain data (Figure 14 insert, Irwin et al. 1991) whereupon a small additional peak ([[arrowup]]) was noted. In order to analyze the hydration process from the standpoint of the polymer (Irwin et al. 1991) a Carr-Purcell-Meiboom-Gill (Farrar 1987) [1]H (60 MHz) NMR experiment on a comparably (e.g., to the uppermost time domain array in Figure 14) hydrated sample, the paramagnetic amide of which had been previously reduced to the N-OH form, is proffered in Figure 15. In this experiment, the semilog plot of normalized resonance areas, as a function of the variable delay, 2[[tau]], clearly demonstrate 2 populations of spin-spin relaxation. The y-intercept for the slowly relaxing H2O population (fast motion) was utilized as a relative measure of the activity of "free" H2O presumably located in an outer hydration shell. The free H2O was found to be only about 2% of the total hydration [1]H2Os. For equilibrium hydrated samples, this value increases to a level >50% whereupon there would be on the order of 10 H2O molecules per monomer unit (Chamulitrat & Irwin 1989).
Figure 16 (Irwin et al. 1991) displays how the "order parameter", 2Azz (Chamulitrat et al. 1988), which we have shown to be related to internal nitroxyl amide reorientation about the macromolecular y axis or main chain, changes as a function of temperature and degree of polymer hydration in spin labeled cell wall pectinic polysaccharides. Both moderately- and fully-hydrated samples (10 and 168 h, respectively) had larger values of 2Azz than the dehydrated control, possibly due to matrix deformation induced by the freezing of bound H2O. However, there is an effect of solvent on Azz which could explain these results. Notwithstanding, the temperature at which the fully-hydrated sample's 2Azz approached the 0 h observation, ca. 256K, was remarkably similar to the temperature (Tmax, Irwin et al. 1985b) where maximal Mn[2+] spin-spin broadening, due to relatively close near neighbors, was observed (Figure 17) for similarly-hydrated CW samples. In this Figure we see that Tmax was not associated with freezing since it was inversely related to the level of bound H2O; however, we did find that Tmax was related to the anionic ligand's structure. Like the Tmax parameter, 2Azz, in the 10 h treatment, approached the dehydrated sample's 2Azz at or above the freezing point of bound water. If this approach to the control (0 h) 2Azz values were due to the thawing of previously-frozen bound H2O, it would occur at a temperature much lower than what was observed, 284K, due to the colligative property of freezing point depression (Daniels & Alberty 1975).
Alkali extraction is the classical chemical method for removing hemicellulose, which is presumably hydrogen bonded to cellulose, from the cell wall (Fry 1986). Unfortunately, such treatment can also break other types of bonds such as glycosidic linkages (via [[beta]]- or trans-elimination; Barrett & Northcote 1965), can hydrolyse methyl esters and acetylated groups as well as modify ionic bonds such as Ca[2+] bridges. The CPMAS/NMR spectrum of a 2 N KOH extracted cell wall matrix (Chamulitrat & Irwin 1989) displayed the loss of the methyl ester's 54 ppm resonance indicating that these were saponified concomitant with the extraction of [[beta]]-D-glucans in hemicellulose. These alkali treatments were performed at room temperature whereupon the [[beta]]-elimination process is less efficient (Barrett & Northcote 1965) therefore we assumed that the most significant effect of the alkali extraction was the removal of hemicellulose as well as the saponification of the methyl ester functional groups. Our EDC-mediated spin labeling reaction was performed after the alkali treatment and EPR spectra were acquired at 77 K as discussed in the previous section. The alkali treated cell wall EPR spectra exhibited a z axis hyperfine coupling, Azz, of 36.79 G which was significantly larger than the Azz for cell walls without the alkali treatment (36.36G for either PME-treated or control CWs). Upon fully hydrating any nitroxyl amide labeled matrix polyuronide, the EPR spectra appear as two overlapping weakly (W ) and strongly (S ) rotationally-immobilized components (Chamulitrat & Irwin 1989). When the nitroxyl amide labeled/alkali treated cell wall sample was hydrated to 85% [w/w] and the EPR spectrum measured (Figure 18 bottom; W/S = 5.03) the W/S ratio was much larger than the enzymatically deesterified (PME-treated, W/S = 3.61) cell wall matrix with a comparable level of hydration. The larger value of W/S in the hemicellulose-removed/demethylated wall matrix relative to that of the specifically deesterified cell walls, indicates that there was an increase in the rotational freedom of the nitroxyl labels by the partial removal of hemicellulose alone. These results suggest that portions of the hemicellulose matrix must be spatially close to the labeled homopolygalacturonan chains. Upon treating the hemicellulose-removed cell walls with Ca[+2] (0.88 cations per binding site; Figure 18 top), the W/S ratio was observed to decrease from 5.0 (Figure 18 bottom) to 3.6. Since the W/S parameter is a reasonable measure of the spin label's rotational freedom, this change in the population of weakly immobilized components, as a function of bound Ca[2+], indicates that Ca[2+] has a strong effect on the mobility of the spin probe and its subtending polymer. These findings are important since virtually all research into the effects of Ca[2+] on living fruit tissue are complicated by the fact that Ca[2+] itself has various, not well understood, physiological effects.
As shown previously (Chamulitrat & Irwin 1989), the behavior of hydrated CW macromolecules can be more extensively investigated when examined with respect to the diffusion of water bound to the CW polymer matrix. Upon increasing the level of hydration, the 2Azz was attenuated and the weakly immobilized nitroxyl amide population increased since the W/S ratio enlarged as a function of bound water. Previously (Chamulitrat et al. 1988) we recognized that there was a collapse of the apparent free volume of the polymer matrix during equilibrium hydration (e.g., as [[upsilon]]1 approaches [[upsilon]]max; the volume fraction of the diluent, [[upsilon]]1 , ~ degree of hydration in units of g H2O/g polymer). Since the translational diffusion of the penetrant, or diluent, molecules in certain polymers (Ferry 1980) is related to free volume theory, it is ostensibly reasonable to consider the nitroxyl amide [[tau]]R's dependence on the H2O of hydration in a similar manner. To this end we have modified the Fujita-Doolittle equation (Ferry 1980, Brown & Sandreczki 1985, Chamulitrat et al. 1988) as
[[Xi]] = - F(1,loge BBC[(F([[tau]]R,[[tau]][ref]DBa8()R ))) = - F(1,loge BBC[(F([{]W/S[}]ref,[{]W/S[}]))) = f2 + BBC{(F(fS( 2, 2),[[upsilon]]1 [[zeta]])) ,
whereupon limitDBA28()SDO9([[upsilon]]1->[[upsilon]]max)DBA8() [[Xi]] = f2 and [[zeta]] = F([f1 - f2],[[[upsilon]]1 - [[upsilon]]1DBa4()[ref]]) .
The terms f2 and [[zeta]] are constants for a given polymer-diluent system corresponding to the free volume fraction in the reference state (f2) and the proportionality constant ([[zeta]] ) for the dependence of the total fractional free volume, f1, on the diluent volume fraction ([[upsilon]]1DBa4()[ref] is the diluent volume fraction in the dehydrated state and is assumed to be zero). We have introduced the W/S ratio (measured at room temperature) to the above equation since we know that, for a decrease in correlation time (faster motion), there is a corresponding increase of the weakly immobilized population or W/S ratio. [[zeta]] can be obtained once the intercept and slope of a plot of - 1/loge [[{]W/S[}]ref/[{]W/S[}]] versus the reciprocal of [[upsilon]]1 is known (Figure 19). The linearity of the plots in Figure 19 indicate that the W/S ratios, relative to the reference or dehydrated state, are good measures of the rotational diffusion of the nitroxyl amide labels which, in turn, correlate (Chamulitrat et al. 1988) with the monomeric translational diffusion coefficient of the diluent bound to the polymer.
The experiments depicted in Figure 19 are evidence that [[zeta]] is a measure of "intermolecular coupling" of the labeled matrix with its surroundings (Chamulitrat & Irwin 1989). [[zeta]]s for the specifically demethylated CW homopolygalacturonans were about 58% lower than those for untreated CW. Interestingly, the PME-treated matrix had a two-fold larger intermolecular coupling constant ([[zeta]] ) than that of the 11% Ca[2+] salt of PGA; this is not surprising since homopolygalacturonan blocks in the wall network are larger (Irwin et al. 1988) than PGA, more complex and therefore should have a greater degree of intermolecular coupling with near neighbor matrix polysaccharides. PGA nitroxyl amide EPR spectra were obtained at room temperature with various levels of hydration as a function of Ca[+2]DBA9()bound whereupon [[zeta]]s increased with increasing the percentage of Ca[2+] bound and saturated ([[zeta]]max [~]DBa7()= 4) at approximately 0.5 bound Ca ions per site. In these studies we have assumed that the intermolecular coupling parameter would display typical saturation behavior with respect to the fraction ([[Phi]]) of binding sites filled with Ca[2+]. To test this idea we have modified the Hill equation (Van Holde 1971)
[[xi]] [=] F( [[zeta]]obs, [[zeta]]max) [=] F( K[[Phi]][n], 1 + K[[Phi]][n])
F( [[xi]], 1 - [[xi]]) [=] K[[Phi]][n]
log10 BBC{(F( [[[xi]], 1 - [[xi]] ])) [=] log10K + n[ ]log10[[Phi]]
whereupon [[zeta]]max was estimated to be approximately 4, K is an arbitrary constant and n, the Hill coefficient, is a measure of a system's cooperativity at equilibrium. The above linearized log-log equation is shown in Figure 19 (insert; n = 2.98 +/- 0.15). In normal, solution phase, usage [[xi]] would be treated as the fractional saturation of an ensemble of binding sites ([[alpha]]DBa8()[--] ) on a macromolecule, [[Phi]] as the total initial concentration of the ligand (AL ) and K as an equilibrium constant. However, if [[zeta]] is a parameter sensitive to the spatial organization of the Ca[2+] cross linkages in the polyuronic acid matrix, then its behavior with respect to [[Phi]], the faction of sites filled with Ca[2+], should be similar to [[alpha]]DBa8()[--] as a function of AL in a cooperative Ca[2+] binding system such as the homopolygalacturonans (Irwin et al. 1984, Irwin et al. 1985). Such behavior argues that [[zeta]] is a parameter which describes the spatial distribution of the Ca[2+] cross linkages; thus, if n ~ 1, the distribution is random whereas if n is much larger than 1, the cations are distributed in a spatially nonrandom or sequential fashion as has been proposed before (Irwin et al. 1984, Irwin et al. 1985) for paramagnetic divalent cations.
SUMMARY
All the experiments which we have presented herein as examples of the spin label technique indicate that these methods can be applied to food systems for the purpose of characterizing perturbations induced by weak intermolecular forces, such as Ca[2+] bridges, on hydration-deformed or dehydrated sugar acid polysaccharides. We have shown the mechanism of covalent attachment of the spin label, 4AT, to acid polysaccharides; we have utilized various types of spectral simulations to calculate [[tau]]R; we have used the temperature dependence of [[tau]]R to determine divergent Eas for different types of molecular motion (local and internal motions); and, lastly, we have delved into the perturbations in the higher order structure and mobility of the sugar acid-containing matrix polysaccharide chains induced by ionic cross bonding as well as other weak forces upon equilibrium hydration.
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FIGURE HEADINGS
Figure 1.
Semilog plot of PGA nitroxyl amide spin concentration as a function of time at ca. 293K. Top inset figure: schematic of a nitroxyl amide spin labeled galacturonan with the magnetic axes, x, y and z, provided. Bottom inset: calibration of the ideal position of a sample within the cavity using a Cr[3+]/Al2O3 crystal (11.38 x 10[15] spins). Various spectral parameters (Azz, Aisotropic), in units of Gauss, were calibrated against the 87.3 G hyperfine splitting of dilute Mn[2+] in a MgO matrix. The parameters, giso or gzz, were calibrated against Cr[3+] in a MgO matrix (gisotropic = 1.9796). The number of nitroxyl spins was calculated against the Cr[3+]/Al2O3 crystal.
Figure 2.
Log-log plot of F([N- OH],[N--ODBA15()[*] ]) as a function of the molar ratio of ascorbate and bound N--ODBA17()[*] before reduction. Prior to reduction, the PGA amide sample had ca. 10% of the carboxyl groups, upon activation with EDC, reacted as the amide.
Figure 3.
Reactions of uronic acids with carbodiimides (see: Hoare & Koshland 1967, Taylor & Conrad 1972).
Figure 4.
Acid uptake in the carbodiimide reaction (used with permission: Taylor & Conrad 1972) with various hydroxy acids at pH 4.75. Reaction conditions: 0.2 mmole of each carboxylic acid and 0.2 mmole of the carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-tolulenesulfonate (CMC), in 20 ml of H2O. As with our 4AT reactions, the pH was maintained at 4.75 by automatic titration with 0.1 M HCl. For this chapter, the original data has been fit to the exponential,
F([H[+]]added,[COOH]) = limDBA15()SDO10(t[->]*) F([H[+]]added,[COOH])[1-exp(-tk)] ,
and iteratively solved for both the tSDO2(*) limit as well as the rate constant, k, whereupon
BBC{(F([A[-]],[AH]))SDO10(obs.) = limDBA15()SDO10(t[->]*) F([H[+]]added,[COOH]) .
In these calculations, we found that k=7.64 h[-1] and 4.65 h[-1] for L-Galactonic and L-Gulonic Acids, respectively; we also established that k=5.6 h[-1] and 4.7 h[-1] for [[beta]]-OH-Butyric and [[gamma]]-OH-Butyric Acids.
Figure 5.
Relationship between the second moment ([H[2]>ave), derived from numerical integrations, and the line width parameter F(d1,d) (defined in the inset figure), whereupon [H[2]>ave was calculated digitally as
[H[2]>ave = F([[Delta]]H,[[Delta]]H[2] ISU(j=1,m, )ISU(i=1,j, ) BBC{(F(dI(H),dH))i) ISU(j=1,m, )ISU(i=1,j, ) (Hj-Ho)[2] BBC{(F(dI(H),dH))i.
In the above empirical relationship, {dI(H)/dH}i represents the relative amplitude of each 1[st] derivative data point, [[Delta]]H the field separation between points and Ho the magnetic field scalar value where the double integral was half maximum (e.g., the center of the spectrum). Used with permission: Irwin et al. 1987.
Figure 6.
Plot of [[kappa]][2] for a PGA-bonded 4AT lattice versus reciprocal [[chi]] (used with permission: Irwin et al. 1988). Inset figure: Dependency of the nitroxyl amide's [H[2]>ave on the molar ratio of spin-dimers to the total anionic ligand monomers ([[chi]]).
Figure 7.
Relationship between covalently bound 4AT and the electron spin-lattice relaxation time-related parameter relative to that predicted for the untreated matrix (control), ascorbate reduced (partial) and aniline reacted (used with permission: Irwin et al. 1987). All ascorbate treatments were performed on the sample represented by the boldly highlighted open square.
Figure 8.
Arrhenius plots of reorientational motion of nitroxyl amide spin labeled PGA using detailed simulations and an empirical expression (used with permission: Chamulitrat et al. 1988).
Figure 9.
Internal motion Eas as a function of the fraction of binding sites occupied by Ca[2+] for 4AT spin labeled PGA and apple CWs (used with permission: Chamulitrat & Irwin 1989).
Figure 10.
CPMAS/NMR spectra of apple CW matrices (0.8 ms of [1]H-[13]C thermal contact; used with permission: Irwin et al. 1988) as a function of N--ODBA17()[*] (top spectrum) or as its reduced, N-OH, form.
Figure 11.
Power saturation plot of transformed F(dI(H),dH) as a function of the square of the microwave perpendicular field, H1DBA4()[2] (in units of G[2]). The original data, F(dI(H),dH), are the 1[st] derivative intensity of the center (m=0) N--ODBA17()[*] peak. We have made the assumption that H1 [[proportional]] R(Pu) (the microwave power output in units of Watts, Poole 1983) and the constant of proportionality does not change from sample to sample (freeze-dried solids). Inset figure: complete power saturation curve and empirical curve fit for both Ca[2+] and control PGA 4AT nitroxyl amides as a function of R(Pu) BBC(in units of mW SUP6(F(1,2))). The empirical curve fit equation used was of the simple biexponential form,
I' = F(I'DBA3()o BBC[(1-eSUP15([BBC{(F(P]R [,P]F[)) -1*{]R(Pu)[/P]R[}])) eSUP15([BBC{(-]R(Pu)[/P]F[)]),1-F(PR,PF)) ,
whereupon I' is {dI(H)/dH}max for each value of R(Pu), I'DBA3()o is the maximum in I' without inhomogeneous broadening while PR and PF are exponential constants in units of R(mW), respectively.
Figure 12.
Partial power saturation curve (Metz et al. 1990) fit for Ca[2+] PGA, control PGA as well as apple CW 4AT nitroxyl amides as a function of Pu (in units of W). The curve fit equation used was the form,
I' = F(Pu [[Psi]],BBc{(1 + [[Gamma]]Pu)[1/2]) F(1, BBc((1+ [[Lambda]]BBc{(1 + [[Gamma]]Pu)[1/2])[2])
where
[[Psi]] = F(IoDBa4()' H1,R(Pu)) , [[Gamma]] = F([[gamma]][2]H1DBa4()[2]T1T2,Pu) and [[Lambda]] = F(T2*,T2) ;
in our calculation we assume H1DBa4()[2] ~ Pu. Making this assumption we find that, for the CW sample, the relaxation parameter, R(T1T2), was 3.21 +/- 0.07 us; the R(T1T2) s for the PGA matrices were 1.91 +/- 0.15 and 1.58 +/- 0.18 us for the Ca[2+] and control treatments, respectively. Inset figure: plot of the relationship between T1 and T2, assuming T2* is constant throughout and that T2* << T2.
Figure 13.
Dependence of polymer hydration on time in a saturated H2O vapor chamber at 293K. Data were fit to an exponential equation; the rate constants, k, are provided in the figure for both CW and PGA.
Figure 14.
Three pulse electron spin-echo modulations of moderately- (top) and fully-hydrated (bottom) PGA nitroxyl amides at 4.2K. The time between the first two pulses was 0.28 us (used with permission: Chamulitrat & Irwin 1989). Inset figure: Fourier transformation of the top experimental data.
Figure 15.
Carr-Purcell-Meiboom-Gill (CPMG; Farrar1987) refocusing spin echo[1]H (60 MHz) NMR T2 experiment on moderately-hydrated PGA nitroxyl amides at 293K.
Figure 16.
Dependence of the EPR nitroxyl amide parameter, 2Azz, on temperature for dehydrated (0 h), moderately-hydrated (10 h) and fully-hydrated (168 h) CW.
Figure 17.
Dependency of Tmax (temperature, in units of K, where maximum Mn[2+]-filled CW linewidths were observed) on CW lattice hydration. Each data point is the mean of four replications (used with permission: Irwin et al. 1985b). Mn[2+] levels varied between 1 x 10[-4] and 3 x 10[-4] moles/g (32-95% of the available sites filled). Bars represent two standard errors of each mean. Inset figure: Temperature dependence of cell wall bound Mn[2+] (6% of the available sites filled) linewidths ([[Delta]]Hpp) in apple CWs.
Figure 18.
EPR Spectra of fully-hydrated spin labeled CW polyuronides (used with permission: Chamulitrat & Irwin 1989). Bottom spectrum: Alkali extracted apple CW spin labeled with 4AT. Top spectrum: Ca[2+]-doped version of the bottom spectrum.
Figure 19.
Fugita-Doolittle plots of variously-treated 4AT spin labeled PGA and CWs (used with permission: Chamulitrat & Irwin 1989). Insert figure: Hill plot showing the dependence of PGA nitroxyl amide [[xi]], derived from Fugita-Doolittle plots, on the fraction of binding sites filled with Ca[2+]. The slope give n = 3 which is evidence for highly positive cooperative Ca[2+] binding (see text).
TABLES
Table 1.
Parameters and Calculated Activation Energies (SE = +/- 0.05 kcal/mol) for Different Models of Rotation.
Brownian 0.3 2.57 x 10[-10] -1.78 0.83 3.91
Diffusion 3.0 3.40 x 10[-9] -1.36 0.63 5.14
Moderate 0.3 6.99 x 10[-10] -1.20 0.55 3.00
Jump 3.0 1.10 x 10[-9] -1.01 0.47 3.46
Strong 0.3 2.46 x 10[-10] -0.59 0.27 1.69
Jump 3.0 3.40 x 10[-9] -0.62 0.28 1.77
Table 2.
Calculated Activation Energies for Nitroxyl Amide Internal Motion Between 233-358K. Eas were calculated from the empirical formula, [[tau]]R = a BBC{(1 - F(Azz'i,Azz'rl))SUP6( b), where a = 6.99 x 10[-10] or 2.57 x 10[-10] and b = -1.2 or -1.78 for moderate jump or Brownian diffusion models, respectively.
PGA 3.37 4.99
Control CW 2.46 3.66
PME 3.57 5.30
