31P NMR quantification and monophasic solvent purification of human and bovine lens phospholipids.

Most lipid extraction procedures [Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497-509; Bligh, E.G., and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification, Can. J. Biochem. Physiol. 37, 911-917] employ biphasic solvent mixtures designed to dissolve the lipids in an organic phase and remove impurities in an aqueous phase. However, when applying these protocols to biological matrices such as that of the ocular lens, the formation of an emulsion layer between the organic and aqueous phases causes poor reproducibility in extraction yields and gives only a small amount of the lipid-containing chloroform phase. In this study, we quantified phospholipids at each step of the Folch et al. extraction protocol and compared the yield of human and bovine lens phospholipids obtained by the Folch-based approach and a novel monophasic methanol extraction method designed to circumvent the problems associated with biphasic extraction protocols. A monophasic methanol extraction coupled with 31P NMR spectroscopy was found to be the simplest, quickest, and most effective method for quantifying the phospholipid content of the lens.

Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin.

The conformational features of dihydrosphingomyelin (DHSM), the major phospholipid of human lens membranes, were investigated by 1H and 31P nuclear magnetic resonance spectroscopy. Several postulates emerge from the observed trends: (a) in partially hydrated samples of DHSM in CDCl3 above 13 mM, at which lipid-lipid interactions prevail, the amide proton is mostly involved in intermolecular H-bonds that link neighboring phospholipids through bridging water molecules. In the absence of water, the NH group is involved in an intramolecular H-bond that restricts the mobility of the phosphate group. (b) In the monomeric form of the lipid molecule, the amide proton of the major conformer is bound intramolecularly with one of the anionic and/or ester oxygens of the phosphate group. A minor conformer may also be present in which the NH proton participates in an intramolecular H-bond linking to the OH group of the sphingoid base. (c) Complete hydration leads to an extension of the head group as water molecules bind to the phosphate and NH groups via H-bonds, thus disrupting the intramolecular H-bonds prevalent at low concentrations.

Conformational studies of sphingolipids by NMR spectroscopy. II. Sphingomyelin.

Sphingomyelin (SM) is the most prevalent sphingolipid in the majority of mammalian membranes. Proton and 31P nuclear magnetic resonance spectral data were acquired to establish the nature of intra- and intermolecular H-bonds in the monomeric and aggregated forms of SM and to assess possible differences between this lipid and dihydrosphingomyelin (DHSM), which lacks the double bond between carbons 4 and 5 of the sphingoid base. The spectral trends suggest the formation of an intramolecular H-bond between the OH group of the sphingosine moiety and the phosphate ester oxygen of the head group. The narrower linewidth and the downfield shift of the resonance corresponding to OH proton in SM suggest that this H-bond is stronger in SM than in DHSM. The NH group appears to be involved predominantly in intramolecular H-bonding in the monomer. As the concentration of SM increases and the molecules come in closer proximity, these intramolecular bonds are partially disrupted and the NH group becomes involved in lipid-water interactions. The difference between the SM and DHSM appears to be not in the nature of these interactions but rather in the degree to which these intermolecular interactions prevail. As SM molecules cannot come as close together as DHSM molecules can, both the NH and OH moieties remain, on average, more intramolecularly bonded as compared to DHSM.

Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter.

PURPOSE: To measure lipid compositional and structural changes in lenses as a result of hyperbaric oxygen (HBO) treatment in vivo. HBO treatment in vivo has been shown to produce increased lens nuclear light scattering. METHODS: Guinea pigs, approximately 650 days old at death, were given 30 and 50 HBO treatments over 10- and 17-week periods, respectively, and the lenses were sectioned into equatorial, cortical, and nuclear regions. Lipid oxidation, composition, and structure were measured using infrared spectroscopy. Phospholipid composition was measured using (31)P-NMR spectroscopy. Data were compared with those obtained from lenses of 29- and 644-day-old untreated guinea pigs. RESULTS: The percentage of sphingolipid approximately doubled with increasing age (29-544 days old). Concomitant with an increase in sphingolipid was an increase in hydrocarbon chain saturation. The extent of normal lens lipid hydrocarbon chain order increased with age from the equatorial and cortical regions to the nucleus. These order data support the hypothesis that the degree of lipid hydrocarbon order is determined by the amount of lipid saturation, as regulated by the content of saturated sphingolipid. Products of lipid oxidation (including lipid hydroxyl, hydroperoxyl, and aldehydes) and lipid disorder increased only in the nuclear region of lenses after 30 HBO treatments, compared with control lenses. Enhanced oxidation correlated with the observed loss of transparency in the central region. HBO treatment in vivo appeared to accelerate age-related changes in lens lipid oxidation, particularly in the nucleus, which possesses less antioxidant capability. CONCLUSIONS: Oxidation could account for the lipid compositional changes that are observed to occur in the lens with age and cataract. Increased lipid oxidation and hydrocarbon chain disorder correlate with increased lens nuclear opacity in the in vivo HBO model.

Alpha-crystallin/lens lipid interactions using resonance energy transfer.

Resonance energy transfer was used to study the interaction of alpha-crystallin with lens cortex lipid vesicles. The binding of alpha-crystallin to cortex lipid vesicles and the preincubation temperature dependence of the binding were confirmed. In this study, the tryptophan of alpha-crystallin was used as the energy donor, and the fluorescence probe N-(5-dimethylaminonaphthalene-1-sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (dansyl DHPE) was chosen as the energy acceptor. Lens cortex lipid vesicles were preincorporated with dansyl DHPE. Energy transfer from the tryptophan of alpha-crystallin to dansyl DHPE was found and the energy transfer efficiency was calculated. There was a higher energy transfer efficiency between alpha-crystallin and dansyl DHPE when alpha-crystallin was preincubated at 65 degrees C compared to 22 degrees C. Data confirmed the binding of alpha-crystallin to lens cortex lipid and showed that alpha-crystallin bound more closely to the surface of cortex vesicles when it was preincubated at a higher temperature. This is probably due to the exposure of hydrophobic surfaces when alpha-crystallin is preincubated at a higher temperature.

Ca(2+)-ATPase activity and lens lipid composition in reconstituted systems.

Lens lipid composition and lipid hydrocarbon chain structure change with age, region and cataract. Since the lens Ca(2+)-ATPase pump is important to the maintenance of calcium homeostasis and lens clarity, muscle sarcoplasmic reticulum Ca(2+)-ATPase was reconstituted with bovine lens lipids and dihydrosphingomyelin, the rare and major phospholipid of the human lens. Ca(2+)-ATPase activity was found to be about 5 times lower when the pump was reconstituted into dihydrosphingomyelin or lens lipids compared to native sarcoplasmic reticulum lipids. The addition of cholesterol to levels ranging from 13-53 mole%, had no affect on reconstituted Ca(2+)-ATPase activity. Ca(2+)-ATPase activity correlated with the degree of hydrocarbon chain saturation. The greater levels of saturation are a consequence of the high sphingolipid content in the reconstituted systems. These data support the hypothesis that changes in lens lipid composition or structure could affect Ca(2+)-ATPase activity in human lenses. Because the mechanisms governing Ca(2+)-ATPase activity in vivo are much more complex than in these simple reconstituted systems, this study represents an initial step in the elucidation of the relationships of endogenous membrane lipid composition-structure and function.

Effect of oxidation on Ca2+ -ATPase activity and membrane lipids in lens epithelial microsomes.

Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. The level of Ca2+ -ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.

Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes.

Membrane lipid composition varies in different tissues and species. Since a defined lipid composition is essential to the function of many membranes, the relationship between membrane lipid composition and structure was determined using infrared and Raman spectroscopy in four membranes containing a calcium pump: rabbit fast and slow twitch muscle sarcoplasmic reticulum and human and bovine lens fiber cell membranes. We found that membrane sphingolipid and phosphatidylcholine content were correlated to a decrease and increase, respectively, in the infrared lipid CH2 symmetric stretching band frequency. We interpret the change in frequency as a change in lipid hydrocarbon chain structural order. This was confirmed by Raman order parameters. The high degree of hydrocarbon chain saturation found in the variable amide chains of sphingolipids is likely to account for this correlation. Lipid phase transition temperature and cooperativity also correlated to sphingolipid and phosphatidylcholine content, and are the forces defining the order in at physiological temperature in the samples studied. Ca(2+)-ATPase caused an increase in the CH2 symmetric stretching frequency in fast twitch muscle sarcoplasmic reticulum (interpreted as an increase in hydrocarbon chain disorder), but had no effect on slow twitch muscle sarcoplasmic reticulum lipid hydrocarbon chain structure. In the natural systems studied, we find that it is the lipid hydrocarbon chain saturation that defines lipid hydrocarbon chain order.

Influence of cholesterol on the interaction of alpha-crystallin with phospholipids.

The influence of cholesterol on the binding of alpha-crystallin to pure phospholipid membranes was studied. The rationale of this investigation stems from two unique aspects of human lens cells: an unusually high level of cholesterol in the membranes and the specific binding of alpha-crystallin to membranes. In the absence of cholesterol, binding of alpha-crystallin liposomes composed of either sphingomyelin, disteroyl-phosphatidylcholine or egg-phosphatidylcholine caused a decrease in the fluorescence intensity and anisotropy of the fluorophore NBD-PE. Since this fluorescence probe resides in the polar headgroup region of the membrane, the observed changes indicated that the binding of alpha-crystallin affected the structure of these membrane regions. The ability of alpha-crystallin to modulate membrane structure suggests yet another potential role for this lens protein. Addition of cholesterol markedly decreased the binding of alpha-crystallin to liposomes composed of either sphingomyelin or disteroylphosphatidylcholine and antagonized the capacity of bound alpha-crystallin to decrease membrane surface order. This antagonism could be explained by the ability of cholesterol to directly decrease the anisotropy of the fluorophore in sphingomyelin membranes unexposed to alpha-crystallin. Thus, with cholesterol present, a further decrease in membrane order upon subsequent binding of alpha-crystallin was less likely. The results obtained with the sphingomyelin liposomes are considered most meaningful, since sphingomyelins are the principal phospholipids in the human lens nuclear membrane and cholesterol preferentially interacts with sphingomyelin. We conclude that cholesterol in lipid membranes can antagonize the binding of alpha-crystallin and thus interfere with the capacity of bound alpha-crystallin to alter membrane order. We suggest that such actions of cholesterol might serve to preserve lens membrane structure in the physiological state where the concentration of soluble alpha-crystallin is great.

Age-related lipid oxidation in human lenses.

PURPOSE: To quantify age-related changes in products of lipid oxidation in human lenses and to relate these changes to membrane hydrocarbon chain structure. Deviation from a well-defined membrane-lipid composition and structure could result in alterations in membrane function and disruption of the homeostasis of the cell. METHODS: Infrared spectroscopy was used to detect lipid compositional and structural changes in human lens membranes associated with age and cataracts. RESULTS: Lipid oxidation increased linearly threefold relative to total phospholipids in subjects ranging in age between 1 and 85 years, as was evident by increases in trans double bonds, lipid carbonyls, and secondary products. There was no statistical difference between the levels of lipid oxidation in the cortex or nucleus. Lipid hydrocarbon chain order (rigidity) increased from approximately 40% at birth to 70% at 80 years of age. Changes in lipid order correlated with changes in the relative content of membrane phosphatidylcholine and sphingomyelin, and with the level of lipid oxidation. CONCLUSIONS: Lipid oxidation increased linearly and uniformly throughout the human lens with age. The change in lipid oxidation with age correlated to a change in lipid order.

Confirmation of the identity of the major phospholipid in human lens membranes.

PURPOSE: To confirm the identity of the major component of the human lens membranes proposed in 1994 to be dihydrosphingomyelin (DH-SPH). METHODS: DH-SPH was prepared by catalytic hydrogenation of the double bond between carbons 4 and 5 of sphingomyelin (SPH). DH-SPH was characterized by phosphorus-31 (31P) and proton (1H) nuclear magnetic resonance (NMR) spectroscopy at different reaction times. The spectroscopic data were compared to those of the major component extracted from human lens membranes. RESULTS: Both the 1H NMR and the 31P NMR spectral resonances of the prepared DH-SPH matched those for the once "unknown phospholipid" that constitutes approximately half the human lens phospholipids. CONCLUSIONS: The match of the spectroscopic NMR data obtained for the DH-SPH prepared by hydrogenation of SPH and those for the major phospholipid isolated from the human lens membranes confirms the identity of this sphingolipid as D-erythro-4,5-dihydrosphingomyelin.

Lipid-protein interactions in human and bovine lens membranes by Fourier transform Raman and infrared spectroscopies.

In other systems, proteins have been shown to alter the molecular structures of lipids in the cell membrane bilayer. We wished to determine if proteins altered the structure of lens lipids. The structure of lipid hydrocarbon chains in urea purified human lens membrane vesicles containing intrinsic, hydrophobically bound proteins was compared to the structure of lipids in vesicles without protein. Fourier transform Raman spectroscopy was used to characterize lipid and protein structure. To study lipid interactions with extrinsic, surface bound proteins, the lipid structure was compared in bovine lipid vesicles with and without alpha-crystallin bound to the surface of the membrane. Lipid structure was studied using Fourier transform infrared spectroscopy. No change in lipid structure was detected even at protein/lipid weight ratios of two to one. Human lens intrinsic proteins contained a high amount of a helical structure (60%), but did not alter hydrocarbon chain interactions.

Thermodynamic phase transition parameters of human lens dihydrosphingomyelin.

Dihydrosphingomyelin (DHS) is the major phospholipid in the human lens. The influence of this phospholipid on membrane structure and function is not known. In this study we used infrared spectroscopy to determine the thermodynamic and molecular structural properties of the hydrocarbon chains of DHS membranes isolated from human lenses. The phase transition temperature of human lens DHS was 9 degrees C higher than for bovine brain sphingomyelin membranes and 14 and 7 degrees C higher than human lens cortical and nuclear membranes, respectively. This increase in the phase transition temperature results in 20% increase in lipid order at 36 degrees C in comparison to that of native membranes and bovine brain sphingomyelin. DHS is likely to provide structural order to the hydrocarbon chain region and upholds the integrity of native membranes under oxidative conditions.

Effect of capillary properties on the sensitivity enhancement in capillary/fiber optical sensors.

The sensitivity enhancement observed in fluorescence signals when a conventional fiber optical sensor is coupled with a quartz or glass capillary results from the partial reflection of the radiation at the sample/internal wall interface and from the internal reflection of the refracted portion within the capillary wall. Thus, the length and absorbing properties of the capillary as well as the nature of the surrounding medium affect the enhancement significantly. To interpret the dramatic changes in enhancement observed experimentally when the absorbing properties of the capillary were changed, a partial reflective waveguide model is reported.

Characterization of a simple Raman capillary/fiber optical sensor.

The characterization of a simple, dual-fiber quartz capillary/fiber optical sensor (C/FOS) for remote excitation and collection of Raman signals is presented. The Raman signals acquired with the C/FOS exhibit a 70-fold sensitivity enhancement and a 50-fold improvement in detectability relative to those obtained with the corresponding conventional dual-fiber sensor without the capillary. A background spectral feature at 790 cm(-)(1) is related to the optical fiber background and is not due to the capillary tube. With no focusing lenses or filters needed at the sample site, the remote Raman C/FOS is easy to assemble and use, and it is relatively inexpensive compared to other designs.

Structural characterization of clear human lens lipid membranes by near-infrared Fourier transform Raman spectroscopy.

Regional differences in human lens membrane lipid composition have been documented and could be responsible for alterations in the function of lens membranes. The phospholipid composition of epithelial membranes of human lenses has been shown to be different from that of fiber membranes. To establish lipid composition-membrane structure relationships, we have examined spectroscopically the structure of lipid membranes from human lens epithelium, cortex and nucleus. Near-infrared Fourier transform Raman spectroscopy was used to obtain the lipid structure of membranes in which the lipid composition was determined previously by 31P-NMR. The disorder (fluidity measured structurally) of the epithelium was evaluated to be 80%, whereas that of the lipids from the cortical and nuclear regions was 55%. The large size of the band at 1650 cm-1 arising from sphingolipids supported the compositional studies which indicate that the major component of human lens membranes is a sphingolipid. Sphingolipids probably account for the high degree of lipid order found in lens membranes. Epithelial membranes were found to contain more glycerolipids and less sphingolipids than fiber cell membranes. This compositional difference would be expected to disorder the epithelial membrane.

Separation and characterization of the unknown phospholipid in human lens membranes.

PURPOSE: The major component of human lens membranes was thought to be sphingomyelin until 1991, when a study by phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy revealed the presence of an unknown phospholipid that constituted approximately half the human lens phospholipids. The objective of this work was to isolate this phospholipid and to elucidate its identity. METHODS: The separation of sphingomyelin from the unknown was accomplished using high-performance liquid chromatography (HPLC) and an amino-bound column. Sphingomyelin standard and the membranes from human lenses were chromatographed. Chromatographic fractions were collected and spectrally characterized by proton (1H) NMR and 31P NMR spectroscopy. RESULTS: The chromatographic method did not affect the integrity of the sphingomyelin. Besides the bands corresponding to the unknown components, the chromatogram of the human lens membranes showed three large peaks, the central one with a shoulder, with elution times similar to that for sphingomyelin. The 1H NMR spectra for the fractions collected during the elution of these peaks showed differences. The study by 31P NMR indicated that the first peak contained the unknown phospholipid. The subsequent fractions showed the presence, in different relative levels, of both the unknown and sphingomyelin. By comparison and interpretation of the two-dimensional 1H NMR spectra for sphingomyelin and for the fraction containing the unknown, the unknown phospholipid is proposed to be 4,5 dihydrosphingomyelin, in which the site of unsaturation present in the sphingosine moiety is no longer present. CONCLUSIONS: The ability to separate the unknown from sphingomyelin and the power of 1H NMR spectroscopy allowed the proposition of the identity of the major component of human lens membranes as 4,5-dihydrosphingomyelin. Although the synthetic compound is known to be involved in the formation of extended hydrogen-bonding networks, its biologic and physicochemical properties need further study.

Regional and age-dependent differences in the phospholipid composition of human lens membranes.

PURPOSE: The long-term purpose of this research was to establish the relationships between composition, structure, and function that affect human lens membranes. The authors hypothesized that the functional differences of epithelial, cortical, and nuclear lens membranes are related to compositional differences. Furthermore, age-dependent alterations in membrane function and structure can also be related to variations in the phospholipid composition. To explore these possibilities, the authors determined the phospholipid composition of epithelial, cortical, and nuclear membranes from pools of human lenses of different ages. METHODS: Membranes were extracted from pools of clear human lenses of different ages using a monophasic methanolic extraction that minimizes the interfacial fluff produced with biphasic extractions. The phospholipid composition was determined by 31P NMR: RESULTS: Only minor differences were detected between cortical and nuclear fractions. All phospholipids, except sphingomyelin, phosphatidylethanolamine, and the phospholipid with a shift of 0.12 parts per million (ppm) in the 31P NMR spectrum, showed significant differences in the epithelial fractions of all age groups compared to the fiber fractions; the percentage of phosphatidylcholine was considerably higher than that in the cortical and nuclear membranes of the same age. Conversely, the percentage of phosphatidylglycerol and lysophosphatidylglycerol was significantly smaller in the epithelial membranes than in the fiber membranes. The age-related changes in the composition of cortical and nuclear membranes were identical. These membranes showed a steady increase with age in the percentage of sphingomyelin and of an unidentified component with a shift of 1.2 ppm. The percentage of phosphatidylcholine decreased with age in both epithelial and fiber membranes. The rate of decrease was greater in the epithelial membranes than in the fiber membranes. Epithelial membranes contained approximately five times more phosphatidylcholine than fiber membranes of corresponding age. CONCLUSION: Regardless of age, the composition of epithelial cell membranes was different than that of cortical and nuclear membranes, which showed similar phospholipid content. This suggests that significant compositional changes occur when epithelial cells become elongated to form fiber cells.

Structural characterization of lipid membranes from clear and cataractous human lenses.

Lipid composition related structural changes in human cataractous lenses was explored by characterizing the hydrophobic hydrocarbon chains in lipid membranes corresponding to twelve Indian cataractous lenses and eight American clear lenses of similar age. The nuclear-lipid phase transitions corresponding to the clear lenses exhibited significantly higher average transition temperatures (nucleus 33 degrees C, cortex 26.3 degrees C) and cooperativities, 38.1, as compared to the value of 24.1 for the cortical-lipid phase transitions. At 36 degrees C, the phase transitions corresponding to cortical and nuclear lipids indicate a similar degree of disorder, 63%, in the hydrocarbon chains, i.e., similar relative amounts of gauche and trans rotomers. The twelve cataractous lenses investigated all had nuclear opacities, four were brunescent and four had cortical opacities. No significant differences were observed in the phase transition parameters (temperature, cooperativity, magnitude, enthalpy) evaluated for the nuclear-lipid membranes corresponding to the different types of cataracts. Furthermore, for the cataractous membranes, the phase transition parameters obtained for the nuclear lipids were comparable to those evaluated for the cortical lipid membranes. However, the cortical lipids exhibited the highest order in membranes from nuclear cataracts without cortical opacity. The cortical lipids from clear, non-cataractous lenses had the lowest level of order. At 36 degrees C, the degree of order in the cortical lipid from clear lenses was comparable to that from nuclear cataractous lenses without cortical opacity. The transition temperature, and cooperativity were significantly higher for cortical lipids from cataractous lenses as compared to those from clear lenses. At 36 degrees C, the degree of order in the cortical lipid membranes was lower for all cataract types vs. clear lens fractions. Our results suggest the possibility that lipid-lipid interactions could be different in cataractous lens membranes. Lipid compositional and chemical differences must account for these altered lipid interactions. These studies will provide a basis for studying lipid-protein interactions and structure-function relationships in the lens membrane.

Estimation of the secondary structure and conformation of bovine lens crystallins by infrared spectroscopy: quantitative analysis and resolution by Fourier self-deconvolution and curve fit.

The secondary structure of six bovine lens protein fractions (two alpha, three beta and one gamma-crystallin) are examined in solution and in solid forms for the first time using FTIR spectroscopy. Films of the nuclear and cortical regions of the bovine lens are also examined. The structure is quantitatively estimated from the vibrational analysis of the resolution-enhanced amide-I profile achieved by Fourier self-deconvolution and linear least-squares curve-fit algorithm. All the protein fractions fold predominantly in a beta-pleated sheet structure with little or no alpha-helical domains in solution or in lyophilized solid form. These proteins also retain their predominant beta-sheet conformation in the cellular phospholipid environment of the lens, in conformity with the structure obtained for all the mammalian species examined to date. Despite structural homology, vibrational data indicate subtle structural differences within each class of the crystallins probably due to presence of several minor substructures/subconformations. Substantial high amounts of turns (approx. 40%) observed in the beta-fractions may have a fundamental implication in stabilizing the tertiary structure of the uniquely folded-proteins vital for the transparency of the lens. These proteins in solid KBr-matrix undergo a major structural change, induced primarily by ionic interactions which refold them in a helical conformation. IR spectroscopy together with band-narrowing procedures has proven to be an effective tool to obtain structural information of proteins in solution, as solid substrates or in a complex biological tissue, such as ocular lens.