A subtransition in a phospholipid with a net charge, dipalmitoylphosphatidylglycerol

Suspensions of dipalmitoylphosphatidylglycerol (DPPG) have been analyzed by differential scanning calorimetry, equilibrium and differential scanning dilatometry, and X-ray diffraction techniques. After the DPPG suspensions are stored several days at 2 degrees C, a new phase transition is observed at a lower temperature than either the main transition or the pretransition. This subtransition has an enthalpy of about 6 kcal/mol and occurs at about 20 degrees C, the exact temperature depending on the buffer used. The lipid partial specific volume increases by 0.035 mL/g upon warming through the subtransition. X-ray diffraction patterns from suspensions in the subgel phase contain orders of a lamellar repeat and several additional sharp and broad wide-angle reflections between 8 and 2 A. As the water content in the specimen is reduced, the lamellar repeat period decreases, whereas the spacings and intensities of these additional wide-angle reflections are unchanged. These data indicate that on incubation at 2 degrees C the lipid molecules crystallize in the plane of each bilayer. X-ray experiments also show that this subgel phase converts to the normal L beta' gel phase above the subtransition.     

Rapid reversible formation of a metastable subgel phase in saturated diacylphosphatidylcholines.

Formation of well ordered lamellar subgel (SGII) phase in aqueous dispersions of L-dipalmitoylphosphatidylcholine upon cooling from the lamellar gel phase, without low-temperature equilibration, is observed in real time using synchrotron x-ray diffraction. It has the same lamellar repeat period as the gel phase from which it was formed but differs in its wide-angle diffraction pattern. The SGII phase forms at about 7 degrees C upon cooling at 2 degrees C/min. In temperature jump experiments at 1 degree C/s from 50 to -5 degrees C, the relaxation time of the lamellar gel-SGII transition is found to be approximately 15 s. The conversion between the lamellar gel and SGII phase is cooperative and rapidly reversible. Upon heating, it coincides in temperature with an endothermic event with a calorimetric enthalpy of 0.35 kcal/mol, the so-called sub-subtransition. Similar sub-subtransitions are also observed calorimetrically at temperatures approximately 10 degrees C below the subtransition, without low-temperature storage, in aqueous dispersions of L-dimyristoylphosphatidylcholine and L-distearoylphosphatidylcholine, but not in racemic DL-dipalmitoylphosphatidylcholine. The formation of the equilibrium lamellar crystalline Lc phase appears to take place only from within the SGII phase.     

Pressure Locking of the Subgel Phase of Hydrated Dipalmitoyl Phosphatidylcholine Bilayers: A Raman Spectroscopic Study

Raman spectra of aqueous dispersions of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) have been measured as a function of pressure (up to 46 kbar) for samples incubated at 2°C and for nonincubated DPPC samples subjected to equally high pressure. The nature of the transition from the GII gel phase of the hydrated lipid into the subgel phase on incubation is entirely different from that of the transition from the GII gel phase into the GIII gel phase of the nonincubated lipid. The GIII gel phase has a monoclinic interchain packing, while the subgel phase exhibits a triclinic interchain structure. It is shown that pressure cannot induce the transition from the GII gel phase to the subgel phase; however, it does stabilize the subgel phase above the subtransition temperature. The mechanism for the formation of the subgel phase and the complex phase behavior of the gel phase of DPPC are rationalized in terms of the dynamic properties of the acyl chains of the lipid molecule.     

Scanning calorimetry reveals a new phase transition in L-alpha-dipalmitoylphosphatidylcholine.

We report a new phase transition in fully hydrated dispersions of dipalmitoylphosphatidylcholine (DPPC). This new transition, called the sub-subtransition, exhibits a transition enthalpy of 0.25 kcal/mol with a Tm at 6.8 degrees C. Unlike the subtransition, no extended low temperature incubation is required to observe the sub-subtransition. This new sub-subgel (SGII) phase may be a precursor to the subgel (SGI) phase, and this discovery is discussed in relation to the current knowledge regarding the polymorphic gel phases of both ester- and ether-linked lipids with identical acyl chains.     

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.     

Kinetics of the subtransition in dipalmitoylphosphatidylcholine

The kinetics of the interconversions of the subgel and gel phases in dipalmitoylphosphatidylcholine have been studied by using differential dilatometry, differential scanning calorimetry (DSC), and neutral buoyancy centrifugation as a function of incubation temperature and deuteriation of the solvent. As seen by others, DSC scans show two peaks in the subgel transition region for incubation temperatures below 1 degree C. After incubation at 0.1 degree C, the DSC peak that occurs at the lower scanning temperature appears with an incubation half-time of 0.5 day and eventually converts into a peak at higher scanning temperature with an incubation half-time of 18 days. By varying the scanning rate, we show that these two peaks merge into one at slow scanning rates with a common equilibrium transition temperature of 13.8 degrees C, in agreement with equilibrium calorimetry and dilatometry (delta V = 0.017 +/- 0.001 mL/g). For incubation temperatures above 4.6 degrees C, only one peak appears in both scanning dilatometry and calorimetry. While the initial rate of subgel conversion is smaller at the higher incubation temperatures, after 300 h a higher percentage of the sample has converted to subgel than at the lower incubation temperatures. We suggest that higher incubation temperatures (near 5 degrees C) are preferable for forming the stable subgel phase, and we present a colliding domain picture that indicates why this may be so. Our results in D2O and the similarity of the kinetics of volume decrease with the kinetics of wide-angle diffraction lines also support the suggestion that the partial loss of interlamellar water plays a kinetic role in subgel formation.     

Polymorphism in myristoylpalmitoylphosphatidylcholine

This study focuses on the mixed-chain lipid myristoylpalmitoylphosphatidylcholine (MPPC) near full hydration. The lipid, synthesized according to the procedure of (Mason et al., 1981a, has a low degree of acyl chain migration. When MPPC is temperature-jumped (T-jumped) from the L alpha phase (T = 38 degrees C) to T = 20 degrees C or below, a subgel phase forms; this formation takes less than 1 h at a temperature below T = 12 degrees C. The subgel remains stable up to T = 29 degrees C. When MPPC is T-jumped from the L alpha phase to T = 24 degrees C or above, a ripple phase forms with coexisting ripple wavelengths of 240 A and 130 A. In contrast, when MPPC is melted from the subgel phase, the ripple phase is characterized by bilayers having a single ripple wavelength of 130 A. In agreement with earlier studies (Stumpel et al., 1983; Serrallach et al., 1984. Structure and thermotropic properties of mixed-chain phosphatidylcholine bilayer membranes. Biochemistry 23:713-720.), no stable gel phase was observed. Instead, an ill-defined low-angle X-ray pattern is initially observed, which gradually transforms into the subgel phase below 20 degrees C, or into the ripple phase above 24 degrees C. In the wide-angle X-ray diffraction, a single peak is observed, similar to the ripple phase wide-angle pattern, that either persists above 24 degrees C or transforms into a multi-peaked subgel wide-angle pattern below 20 degrees C. The absence of a gel phase can be understood phenomenologically as the relative dominance of the subgel phase in mixed-chain PCs compared to same-chain PCs. The subgel structure and molecular interactions responsible for this comparative behavior are interesting open issues.     

Phospholipids chiral at phosphorus. Characterization of the sub-gel phase of thiophosphatidylcholines by use of X-ray diffraction, phosphorus-31 nuclear magnetic resonance, and Fourier transform infrared spectroscopy

A recent study using differential scanning calorimetry (DSC) showed that the thermotropic phase behavior of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) is sensitive to the configuration at phosphorus and that the Rp isomer displayed only a broad transition at 45.6 degrees C [Wisner, D. A., Rosario-Jansen, T., & Tsai, M.-D. (1986) J. Am. Chem. Soc. 108, 8064-8068]. We have employed X-ray diffraction, 31P NMR, and Fourier transform infrared (FT-IR) spectroscopy to characterize various phases of the isomers of DPPsC, to compare the structural differences between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and isomers of DPPsC, and to identify structural factors responsible for the unique behavior of the RP isomer. The results from all three techniques support the previous proposal based on DSC studies that (SP)- and (RP + SP)-DPPsC undergo a subtransition, a pretransition, and a main transition analogous to those of DPPC, while (RP)-DPPsC is quite stable at the subgel phase and undergoes a direct subgel----liquid-crystalline transition at 46 degrees C. Quantitative differences between DPPC and DPPsC (i.e., the effect of sulfur substitution rather than the configurational effect) in the subgel phase have also been observed in the chain spacing, the motional averaging, and the factor group splitting (revealed by X-ray diffraction, 31P NMR, and FT-IR, respectively). In particular, DPPsC isomers are motionally rigid and show enhanced factor group splitting in the subgel phase. These results suggest that DPPsC is packed in different subcells relative to DPPC in the subgel phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of L-arginine with dihexadecylphosphate unilamellar liposomes: the effect of the lipid phase organization

The interaction of L-arginine with unilamellar liposomes of dihexadecylphosphate sodium salt (DHP-Na) has been investigated using calorimetric, light scattering, fluorescence spectroscopy and zeta-potential techniques. Heating from room temperature, the bilayer exhibits a phase transition from a subgel (L(c)) to the gel (L(beta')) phase as well as a pre-transition (L(beta')-P(beta')), which is followed by the main lipid phase transition (P(beta')-L(alpha)). Direct studies of the interaction of L-arginine with the DHP-Na bilayers via isothermal titration calorimetry at 27 degrees C depict significant differences between samples in the L(c) and the L(beta') phases reflecting the effect of molecular organization of the lipids upon the interaction. While L-arginine has only a small impact upon the L(c) to L(beta') phase transition, it affects more significantly the transition temperature as well as the shape of the DSC peaks of the main lipid phase transition. Based on fluorescence and zeta-potential studies, the permeability of L-arginine through the liposomal membrane is higher within the temperature range of the main lipid phase transition. Encapsulated l-arginine obstructs the formation of the subgel phase.     

Effect of subtransition in 1,2-dipalmitoylphosphatidylcholine model membrane on the spin probe motion

The effect of subgel----gel phase transition (subtransition) on conventional electron spin resonance spectra of cholestane, fatty acid and alkylammonium-type spin probes has been studied in aqueous 1,2-dipalmitoyl-sn-phosphatidylcholine dispersions. The cooperative onset of the cholestane spin probe rotation about its long axis, with an effective correlation time of 2-3 ns, has been detected at a temperature coinciding with the calorimetric substransition, indicating onset of the host lipid rotational motion. The lipid rotation results in dissolution of the spin probe clusters in the host lipid. In the gel phase, the lateral distribution of impurity molecules is more isotropic than in the subgel phase.     

Comparison of the effects of NaCl on the thermotropic behaviour of sn-1' and sn-3' stereoisomers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol

The phase behaviour of liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG) and the corresponding sn-3' stereoisomer (3'-DMPG) were studied by DSC as a function of NaCl concentration. The melting of the metastable gel phase to the liquid-crystalline phase was similar for both lipids. However, in the presence of salt and at 6 degrees C (T less than Tp) the gel phase of both stereoisomers of DMPG was shown to be metastable and a new phase nominated here as the highly crystalline phase was formed as the stable state. However, significant differences in the formation and melting of the highly crystalline phase were evident between the two polar headgroup stereoisomers. For 3'-DMPG in the presence of 300 mM NaCl the melting enthalpy of this phase is approx. 82 kJ/mol and the transition temperature about 11 degrees higher (at 33.6 degrees C) than for the gel to liquid-crystalline phase transition (25 kJ/mol at 23.0 degrees C). In the presence of 0.15-1.2 M NaCl at 6 to 10 degrees C the formation of the highly crystalline phase of 3'-DMPG is complete within 2 to 5 days, increasing [NaCl] facilitates the rate. For a 1:1 mixture of 1'- and 3'-DMPG the formation of the highly crystalline phase requires several weeks and melts at about 20 degrees higher than the gel phase (at approx. 40 degrees C). For 1'-DMPG partial conversion into the highly crystalline phase requires several months. For 3'-DMPG several intermediate phases appeared as endothermic peaks between the main phase transition temperature and the melting temperature of the highly crystalline phase. In contrast, for 1'-DMPG and the 1:1 mixture the subgel phase appears to be the only metastable intermediate phase. Different monovalent cations differ in their effect on the metastable behaviour.     

Kinetics for the subgel phase formation in DPPC/DOPC mixed bilayers

We analyzed the kinetics for the subgel (SGI) phase formation in DPPC/DOPC binary bilayers paying attention to DOPC-induced modification of the bilayer physical properties. Differential scanning calorimetry and X-ray diffraction revealed that addition of DOPC reduced the apparent initial lag time to start the SGI phase formation, and that the SGI phase in the binary bilayers had basically the same structure as that in pure DPPC bilayers though addition of DOPC markedly increased the peak temperature and enthalpy of the subtransition in heating. Moreover, addition of DOPC abolished the prolongation of the initial lag time in pure DPPC bilayers induced by lowering the incubation temperature from 0 to ?5 °C. Our results suggested that DOPC molecules work as a diffusion enhancer to promote the nucleation of the SGI phase, and relatively destabilize the gel phase so that the formed SGI phase transforms into the ripple phase in heating.     

Mechanism and kinetics of the subtransition in hydrated L-dipalmitoylphosphatidylcholine

The mechanism of the subtransitions (Lc to L beta') in L-dipalmitoylphosphatidylcholine bilayers in excess water has been investigated by time-resolved X-ray diffraction using synchrotron radiation. The temperature dependence of the diffraction patterns closely correlate with the asymmetric excess specific heat variation recorded by differential scanning calorimetry. During the subtransition two prominent wide-angle reflections, characteristic of the low-temperature crystalline phase, Lc, gradually change such that a sharp peak at a spacing of 0.430 nm decreases in intensity and ultimately disappears while a broader peak initially located at 0.375 nm progressively shifts to an eventual spacing of 0.410 nm. This behaviour is interpreted as a lateral deformation of the acyl chain packing subcell as the chains begin to rotate until a state is reached where the chains pack on a regular hexagonal array characteristic of the L beta phase. An increase in lamellar repeat distance from 6.0 to 6.4 nm takes place simultaneously with the acyl chain rearrangement at relatively low (5 K/min) as well as high (6 K/s) heating rates. As judged from the shape of the wide-angle peak, transformation to L beta' phase occurs some minutes after transition to the L beta phase. The X-ray data characterise the subtransition as a continuous (second order) phase transition in which a presumably orthorhombic subcell is transformed into a hexagonal subcell in a gradual process. In temperature jump experiments at 6 K/s between 0 degree C and 80 degrees C the relaxation time of the subtransition was found to be about 5 s while the relaxation time of the main gel to liquid-crystalline transition was about 2 s.     

Comparative study of the gel phases of ether- and ester-linked phosphatidylcholines

Calorimetric, X-ray diffraction, and 31P nuclear magnetic resonance (NMR) studies of aqueous dispersions of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) gel phases at low temperatures (-60 to 22 degrees C) show thermal, structural, and dynamic differences when compared to aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) gel phases at corresponding temperatures. Differential scanning calorimetry of DHPC dispersions demonstrates a reversible, low-enthalpy "subtransition" at 4 degrees C in contrast to the conditionally reversible, high-enthalpy subtransition observed at 17 degrees C for annealed DPPC bilayers. X-ray diffraction studies indicate that DHPC dispersions form a lamellar gel phase with dav congruent to 46 A both above and below the "subtransition". It is suggested that the reduced dav observed for DHPC (46 A as compared to 64 A in DPPC) is due to an interdigitated lamellar gel phase which exists at all temperatures below the pretransition at 35 degrees C. 31P NMR spectra of DHPC gel-phase bilayers show an axially symmetric chemical shift anisotropy powder pattern which remains sharp down to -20 degrees C, suggesting the presence of fast axial diffusion. In contrast, 31P spectra of DPPC bilayers indicate this type of motion is frozen out at approximately 0 degrees C.     

A differential scanning calorimetric study of the thermotropic phase behavior of model membranes composed of phosphatidylcholines containing linear saturated fatty acyl chains

The thermotropic phase behavior of a series of 1,2-diacylphosphatidylcholines containing linear saturated acyl chains of 10-22 carbons was studied by differential scanning calorimetry. When fully hydrated and thoroughly equilibrated by prolonged incubation at appropriate low temperatures, all of the compounds studied form an apparently stable subgel phase (the Lc phase). The formation of the stable Lc phase is a complex process which apparently proceeds via a number of metastable intermediates after being nucleated by incubation at appropriate low temperatures. The process of Lc phase formation is subject to considerable hysteresis, and our observations indicate that the kinetic limitations become more severe as the length of the acyl chain increases. The kinetics of Lc phase formation also depend upon whether the acyl chains contain an odd or an even number of carbon atoms. The Lc phase is unstable at higher temperatures and upon heating converts to the so-called liquid-crystalline state (the L alpha phase). The conversion from the stable Lc to the L alpha phase can be a direct, albeit a multistage process, as observed with very short chain phosphatidylcholines, or one or more stable gel states may exist between the Lc and L alpha states. For the longer chain compounds, conversions from one stable gel phase to another become separated on the temperature scale, so that discrete subtransition, pretransition, and gel/liquid-crystalline phase transition events are observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal phase behaviour and structure of hydrated mixtures between dipalmitoyl- and dihexadecylphosphatidylcholine

Mixtures of 1,2-dipalmitoyl- and 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC and DHPC) in dispersion with excess water were studied by differential scanning calorimetry (DSC) and X-ray diffraction techniques. The transition parameters of the main gel-to-liquid crystalline transition show a monotonous dependence on the composition, indicating ideal miscibility of the two lipids, in keeping with the closely similar structures of the pure, hydrated lipids in the P beta' and L alpha states. The pre-transition shows a depression to a minimum temperature of 23 degrees C occurring around equimolar mixtures. Below the pre-transition temperatures, the L beta' gel phase of DPPC maintains bimolecular structure up to DHPC admixtures of 50 mol%, with adaptations in hydrocarbon chain packing and multilayer periodicity. On the side of DHPC, the interdigitated gel structure shows full solubility for DPPC up to equimolarity without major structural changes. The crystalline Lc-phase of DPPC exhibits immiscibility with DHPC, demonstrated by the fact that the subtransition is abolished already at less than 15 mol% DHPC. DHPC, below its subtransition, can accommodate up to 50 mol% DPPC within an interdigitated layer structure with unperturbed, crystalline hydrocarbon chain packing.     

Effects of very small amounts of cholesterol on gel-phase phosphatidylcholine membranes

The mobility of 5-doxyl stearic acid spin label (5-SASL) in the gel phase of dipalmitoylphosphatidylcholine membranes between the main transition and subtransition temperatures was studied as a function of cholesterol content. Very small amounts of cholesterol (0.01-1 mol%) cause a dramatic increase in the mobility of 5-SASL. Temperature-drop experiments from 38 degrees C to 28 degrees C were made across the pretransition temperature and the rate of approach to equilibrium was measured. Cholesterol at low concentrations also affects this rate. The membrane reached equilibrium after 10 h in the absence of cholesterol, 3 h at 0.01 mol% cholesterol, and less than 10 min at 0.03 mol% cholesterol.     

X-ray structure study of thermotropic phases in isoacylphosphatidylcholine multibilayers.

Structures of lamellar phases in aqueous dispersions of diisoacylphosphatidylcholines (17iPC and 20iPC) were determined by x-ray diffraction methods. In agreement with previous DSC studies, subgel, gel, and liquid crystal phases were observed in each homolog. The subgel Lc(c') phases of both homologs show significant two-dimensional long range order and can be described by rectangular lattices. The dimensions of the two rectangular unit cells differ in that the side chains are canted (approximately 33 degrees) in the 20iPC homolog, while in 17iPC the side chains are normal to the bilayer plane. The gel L beta phases of 17iPC (Tgg = 17-19.5 degrees C) and 20iPC (Tgg = 44 degrees C) are similar but not identical and are consistent with a distorted, pseudohexagonal lattice for the rotationally disordered side chains. The liquid crystal phases of 17iPC (Tgl = 28 degrees C) and 20iPC (Tgl = 52 degrees C) are structurally similar and are typical of lipids with fluid side chains. Significant but different changes occur in the long spacings at Tgg and Tgl for the two homologs. This implies that interfacial states (particularly in the subgel phases) differ in the two homologs below the liquid crystal phase transition temperature.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 degrees C/min while the ice nucleation temperature was varied between -3 and -10 degrees C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below -4 degrees C and cell survival exhibits an optimum at a nucleation temperature of -6 degrees C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at -3 degrees C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at -10 degrees C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in alpha-helical structures and a concomitant increase in beta-sheet structures starting at an onset temperature of approximately 48 degrees C.     

Metastability and polymorphism in the gel phase of 1,2-dipalmitoyl-3-SN-phosphatidylcholine. A Fourier transform infrared study of the subtransition.

Fourier transform infrared spectroscopy was used to study the metastability of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) at temperatures near 0 degrees C. It was found that when DPPC is incubated at 2 degrees C for three days the two-dimensional acyl chain packing changes from one resulting in spectra typical of an orthorhombic subcell to one resembling that found in triclinically packed acyl systems. This transition proceeds in two stages. The first step, requiring less than one day, approximates first-order kinetics; the second stage proceeds with second- or higher-order kinetics. Comparison of spectra recorded at -36 degrees C with and without prior incubation at 2 degrees C shows that there are two stable low temperature forms of DPPC; that is, DPPC is metastable only within a narrow temperature range. A study of the thermotropic behavior in the range 0-45 degrees C shows that the subtransition near 15 degrees C is a transition from the alternate form to one with orthorhombic characteristics. Spectral changes at the pretransition and the main phase transition demonstrate that there are differences in behavior that are related to the thermal history of the sample.