Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.     

Infrared absorption study of the heme pocket dynamics of carbonmonoxyheme proteins

The temperature dependencies of the infrared absorption CO bands of carboxy complexes of horseradish peroxidase (HRP(CO)) in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape. The bands' anharmonic behavior is explained assuming that there is a higher-energy set of conformational substates (CSS(h)), which are populated upon heating and correspond to the protein substates with disordered water molecules in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA), shows that the low energy CSS(h) exists also in the open conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the HRP(CO)+BHA and closed conformations of Mb(CO) and Hb(CO), the heme pocket is packed with BHA and different amino acids, the CSS(h) has much higher energy and is hardly populated even at the highest temperatures. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature-independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This oscillator can be a moving water molecule locked in the heme pocket in the whole interval of temperatures or a trehalose molecule located in the heme pocket.     

Solvent dependent and independent motions of CO-horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.     

Conformational substates and dynamic properties of carbonmonoxy hemoglobin

Heme pocket dynamics of human carbonmonoxy hemoglobin (HbCO) is studied by Fourier transform infrared spectroscopy. The CO stretching band at various temperatures in the interval 300-10 K is analyzed in terms of three taxonomic A substates; however, in HbCO the band attributed to the A(1) taxonomic substate accounts for approximately 90% of the total intensity in the pH range 8.8-4.5. Two different regimes as a function of temperature are observed: below 160 K, the peak frequency and the bandwidth of the A(1) band have constant values whereas, above this temperature, a linear temperature dependence is observed, suggesting the occurrence of transitions between statistical substates within the A(1) taxonomic substate in this protein. The relationship between the heme pocket dynamics (as monitored by the thermal behavior of the CO stretching band), the overall dynamic properties of the protein matrix (as monitored by the thermal behavior of Amide II and Amide I' bands) and the glass transition of the solvent (as monitored by the thermal behavior of the bending band of water) is also investigated. From this analysis, we derive the picture of a very soft heme pocket of hemoglobin characterized by rather large anharmonic terms and strongly coupled to the dynamic properties of the solvent.     

海藻糖对脂肪酶的保护机理及酶失活动力学

采用自制的磁性固定化酶(MIE),考察了高温下二糖类对酶的保护作用。结果显示:海藻糖对悬浮于水溶液中的MIE没有保护作用;而在高温干燥后,对酶的保护作用效果依次为:海藻糖>乳糖>蔗糖,支持‘玻璃态学说’;此外,采用两步失活动力学模型能够较好的拟合酶的失活过程,并且得到酶的失活速率常数k和半衰期t1/2,加入海藻糖和乳糖之后,MIE的半衰期分别增长了31和23倍。     

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

Carbonmonoxy horseradish peroxidase as a function of pH and substrate: influence of local electric fields on the optical and infrared spectra

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6.0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrational stretch frequency is at 1903 cm(-1). Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of approximately 320 cm(-1) in the Q(0,0) absorption band that is clearly evident at < 100 K and shifts the CO absorption to 1916 cm(-1). Increasing the pH to 9.3 also causes a split in the Q(0,0) optical band and elicits a shift in nu(CO) to a higher frequency (1936 cm(-1)). The splitting of the Q(0,0) band and the shifts in the IR spectra are both consistent with changes in the local electric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the distal histidine, although other effects are also considered. The larger effect on the Q(0,0) band with substrate at low pH and the shift of nu(CO) at high pH can be rationalized by the directionality of the field and the orientation dependence of dipolar interactions.     

Iron-histidine resonance raman band of deoxyheme proteins: effects of anharmonic coupling and glass-liquid phase transition

Weak anharmonic coupling of two soft molecular vibrations is shown to cause pronounced temperature dependence of the corresponding resonance Raman bands. The developed theory is used to interpret the temperature dependence of the iron-histidine band of deoxyheme proteins and model compounds. It is shown that anharmonic coupling of the iron-histidine and heme doming vibrations must cause pronounced broadening of the band, its asymmetry, and shift of its maximum to the red upon heating. It also can lead to a structured shape of this band at room temperature. Proper consideration of the anharmonic coupling allows simulation of the temperature dependence of the iron-histidine band shape of horse heart myoglobin in the temperature interval of 10-300 K, using the minimum number of necessary parameters. Analysis of this temperature dependence clearly shows that the iron-histidine band of deoxyheme proteins is sensitive to the glass-liquid phase transition in the protein hydration shell, which takes place at 160-190 K.     

Observation of the FeIV=O stretching Raman band for a thiolate-ligated heme protein. Compound I of chloroperoxidase.

The FeIV=O stretching vibration has never been identified for a cysteine-coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed-flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm-1, which shifted to 756 cm-1 with the 18O derivative, but the v4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin pi cation radical. The second intermediate gave an intense v4 band at 1,372 cm-1 but no oxygen isotope-sensitive Raman band, suggesting oxygen exchange with bulk water.     

Local dynamic properties of the heme pocket in native and solvent-induced molten-globule-like states of cytochrome c

We report the Soret absorption band, down to cryogenic temperature, of native and molten-globule-like state of horse heart cytochrome c. The band profile is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Franck-Condon approximation. From the temperature dependence of the Gaussian broadening and of the peak position, we obtain information on the 'bath' of low frequency harmonic motions of the heme group within the heme pocket. The reported data indicate that, compared to the native state, the less rigid tertiary structure of the molten globule is reflected in a higher flexibility of the heme pocket and in greater conformational disorder, allowing the transduction of large-amplitude motion of the protein to the dynamics of the heme pocket.     

Combined effects of trehalose and cations on the thermal resistance of beta-galactosidase in freeze-dried systems

The purpose of this study was to investigate the combined effects of trehalose and cations on the preservation of beta-galactosidase in freeze-dried systems and their relationship to physical properties. Differential scanning calorimetry was employed to measure the glass transition temperature (T(g)) and the endothermal peak area, related to the amount of crystalline trehalose dihydrate present in the samples. In systems in which the trehalose matrix was humidified to conditions which allowed a high proportion of trehalose to crystallize, the enzyme was rapidly inactivated upon heating at 70 degrees C. In these conditions the addition of CsCl, NaCl and particularly KCl or MgCl(2), improved the enzyme stability with respect to that observed in matrices containing only trehalose. For a given moisture content, addition of salts produced very little change on the glass transition temperature; therefore the protective effect could not be attributed to a higher T(g) value. The crystallization of trehalose dihydrate in the humidified samples was delayed in the trehalose/salt systems (principally in the presence of Mg(2+)) and a parallel improvement of enzyme stability was observed.     

Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.     

Interaction of bd-type quinol oxidase from Escherichia coli and carbon monoxide: Heme d binds CO with high affinity

Comparative studies on the interaction of the membrane-bound and detergent-solubilized forms of the enzyme in the fully reduced state with carbon monoxide at room temperature have been carried out. CO brings about a bathochromic shift of the heme d band with a maximum at 644 nm and a minimum at 624 nm, and a peak at 540 nm. In the Soret band, CO binding to cytochrome bd results in absorption decrease and minima at 430 and 445 nm. Absorption perturbations in the Soret band and at 540 nm occur in parallel with the changes at 630 nm and reach saturation at 3-5 microM CO. The peak at 540 nm is probably either beta-band of the heme d-CO complex or part of its split alpha-band. In both forms of cytochrome bd, CO reacts predominantly with heme d. Addition of high CO concentrations to the solubilized cytochrome bd results in additional spectral changes in the gamma-band attributable to the reaction of the ligand with 10-15% of low-spin heme b558. High-spin heme b595 does not bind CO even at high concentrations of the ligand. The apparent dissociation constant values for the heme d-CO complex of the membrane-bound and detergent-solubilized forms of the fully reduced enzyme are about 70 and 80 nM, respectively.     

Ligand, cofactor, and residue vibrations in the catalytic site of endothelial nitric oxide synthase

A study of bovine endothelial nitric oxide synthase by Fourier transform infrared (FTIR) spectroscopy in the 1000-2500 cm(-)(1) range is reported. Binding of CO to the reduced enzyme gives two heme(II)-CO nu(C)(-)(O) stretches (1927 and 1904 cm(-)(1)) which appear to be in rapid equilibrium. Photolysis of this heme(II)-CO compound is accompanied by perturbation of the local fine structure around the catalytic site giving vibrational changes of protein backbone, substrate, amino acid residues, and cofactors, to which heme, substrate arginine, and catalytic site residues contribute. Possible assignments of vibrations to heme, substrate arginine, and catalytic site residues are discussed. The discussion of assignments is informed by known structures, absorbance frequencies, and extinction coefficients of residues and cofactors, analysis of H(2)O-D(2)O exchange effects, analysis of substrate (14)N-(15)N (guanidinium)-arginine exchange effects, and comparison with the nNOS isoform (which differs in the replacement of asparagine 368 with an aspartate within the substrate binding site). The FTIR data can be modeled on the known structure of the catalytic site and indicate the extent of modulation of vibrational modes upon photolysis of the CO compound.     

Correct interpretation of heme protein spectra allows distinguishing between the heme and the protein dynamics

It is shown by using the vibronic approach that the iron displacement out of the porphyrin plane in deoxyheme proteins intermixes the porphyrin pi and axial iron-histidine sigma electronic subsystems. This intermixing explains the substantial coupling of the iron-histidine vibration to the heme Soret excitation, the appearance of the iron-histidine band in the corresponding resonance Raman spectra, and a number of other experimental data, including the dependence of the iron-histidine vibrational frequency on the extent of the iron displacement out of the porphyrin plane. This dependence implies that there is an anharmonic coupling between the corresponding vibrations, which is shown to be the cause of the specific temperature dependence of the iron-histidine band. The anharmonic coupling and the dependence of the dipole transition moment of the charge transfer optical absorption band III on the iron-porphyrin distance cause the anomalous temperature and pressure dependencies of this band. It is shown that the change in both the magnitude and the distribution of the iron-porphyrin distance is expected to affect the band III intensity. Consequently, the stationarity of the band III intensity can be considered as a signature of the stationarity of the iron-porphyrin distance and its distribution in deoxyheme proteins, whereas the band III position and width could be also affected by the change in the protein electric field, caused by the protein globule dynamics.     

The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.     

Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Molecular dynamics simulation of sucrose- and trehalose-coated carboxy-myoglobin

We performed a room temperature molecular dynamics (MD) simulation on a system containing 1 carboxy-myoglobin (MbCO) molecule in a sucrose-water matrix of identical composition (89% [sucrose/(sucrose + water)] w/w) as for a previous trehalose-water-MbCO simulation (Cottone et al., Biophys J 2001;80:931-938). Results show that, as for trehalose, the amplitude of protein atomic mean-square fluctuations, on the nanosecond timescale, is reduced with respect to aqueous solutions also in sucrose. A detailed comparison as a function of residue number evidences mobility differences along the protein backbone, which can be related to a different efficacy in bioprotection. Different heme pocket structures are observed in the 2 systems. The joint distribution of the magnitude of the electric field at the CO oxygen atom and of the angle between the field and the CO unit vector shows a secondary maximum in sucrose, absent in trehalose. This can explain the CO stretching band profile (A substates distribution) differences evidenced by infrared spectroscopy in sucrose- and trehalose-coated MbCO (Giuffrida et al., J Phys Chem B 2004;108:15415-15421), and in particular the appearance of a further substate in sucrose. Analysis of hydrogen bonds at the protein-solvent interface shows that the fraction of water molecules shared between the protein and the sugar is lower in sucrose than in trehalose, in spite of a larger number of water molecules bound to the protein in the former system, thus indicating a lower protein-matrix coupling, as recently observed by Fourier transform infrared (FTIR) experiments (Giuffrida et al., J Phys Chem B 2004;108:15415-15421).     

A Calorimetric Study of the Glass Transition Behaviors in Axes of Bean Seeds with Relevance to Storage Stability

Although the presence of intracellular aqueous glasses has been established in seeds, their physiological role in storage stability is still conjectural. Therefore, we examined, using differential scanning calorimetry, the thermal behavior of glass transitions in axes of bean (Phaseolus vulgaris L.) with water contents (WC) between 0 and 1 g H2O/g dry weight (g/g) and temperatures between -120 and +120[deg]C. Three types of thermal behaviors associated with the glass transition were observed. The appearance, the glass -> liquid transition temperature, and the amount of energy released during these transitions were dependent on the tissue WC. No glass transitions were observed at WC lower than 0.03 and higher than 0.45 g/g. A brief exposure to 100[deg]C altered the glass properties of tissues with WC between 0.03 and 0.08 g/g but did not affect the thermal behavior of glasses with higher WC, demonstrating that thermal history is important to the intracellular glass behavior at lower WC. Correspondence of data from bean to models predicting the effects of glass components on the glass -> liquid transition temperature suggests that the intracellular glasses are composed of a highly complex sugar matrix, in which sugar and water molecules interact together and influence the glass properties. Our data provide evidence that additional glass properties must be characterized to understand the implications of a glassy state in storage stability of seeds.