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Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

The possibility of performing FT-IR spectromicroscopy experiments on individual living cells is the focus of considerable attention. Among the applications of interest, the obtainment of structural information in rapid measurements, with a time resolution of the minute or better, is a prized goal. In this work, we show that the use of synchrotron FT-IR spectromicroscopy allows one to extract weak spectral changes, of less than 10(-3) au per minute, in the absorption spectrum of single rod cells following photostimulation. We also show that absorption changes are accompanied by other optical effects due to changes in the real part of the refractive index of the cell. The use of two-dimensional correlation spectroscopy allows us to assign bands to specific molecular chromophores and to extract weak spectral variations in the presence of a noisy background.

Over the years Fourier-Transform Infrared (FTIR) spectroscopy has been widely employed in the structural and functional characterization of biomolecules. The introduction of infrared (IR) microscopes and of synchrotron light sources has created expectations that FTIR could become a generally viable technique to study both structure and reactivity in vivo, inside single cells, by performing measurements that up to a few years ago were the preserve of in vitro experiments on purified macromolecules. In this review we present the state-of-the-art in the application of FTIR spectromicroscopy as a technique for the study of structure and dynamics in single cells, we discuss the performance requirements for this application and review developments in sample handling methods.

We demonstrate the capability of synchrotron-based Fourier-Transform Infrared spectromicroscopy to detect metabolite formation in single, living cells of the unicellular algae Chlamydomonas reinhardtii. We show that the high brightness of the source provides a sufficient signal-to-noise ratio to detect small molecular species accumulating in a spot about 15 microm in size. Time resolved measurements are carried out on cells grown heterotrophically under low-light conditions to study the evolution of products of anaerobic metabolism. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

The objective of this exploratory study was to evaluate the feasibility of using Fourier-Transform Infrared (FTIR) spectromicroscopy to characterize formalin-fixed, paraffin-embedded human esophageal tissues. Matched histologically normal esophageal squamous epithelium (NS), premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC) tissues, each defined according to strict clinicopathologic criteria, were obtained from patients who underwent esophageal resection. Using confocal IR microscopy, measurements in the mid-IR spectral region were carried out in transflection configuration, scanning regions of interest in 15 microm steps. A multidimensional dataset reporting the spectroscopic properties at each sampled point were analyzed by performing a hierarchical cluster analysis on the second derivative of spectral traces. Normal esophageal epithelia were characterized by a few well defined regions, mostly of large size (tens of contiguous pixels), which correlated with tissue histology, specifically the basal cell layer. BE tissues had characteristic regions localized to gland crypts, ranging in size from one pixel to a few tens of pixels, which displayed IR spectra with defined absorption features characteristic of glycoproteins. The incorporation of synchrotron light to improve the resolution of individual cells in BE tissues has demonstrated that these glycoproteins are associated with goblet cells, the characteristic cell type defining BE. Whereas the highly fragmented regions identified in EADC likely reflect tumor heterogeneity, FTIR mapping would appear to be a potentially useful technique to identify premalignant BE tissues. The technical feasibility of using FTIR to characterize formalin-fixed, paraffin-embedded human esophageal tissues demonstrates the potential of this technique to study archival human BE tissue specimens via automated screening techniques.

We have recorded nanoscale topography and infrared chemical fingerprints of attomole layered lipids consisting of dimyristoylpho-sphatidylcholine on silicon and mica. Lipids deposited on mica built stacks consisting of up to 25 bilayers, each approximately 5 nm thick, spanning a range from 5-125 nm in height. Contrast evaluation as a function of layer thickness provides the near-field depth resolution.

The phase behavior of binary mixtures of egg sphingomyelin and cholesterol has been inspected by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in the amide I' band region of the spectrum. Because cholesterol does not have any major absorption bands in this region, effects seen in the spectra of mixtures of sphingomyelin and cholesterol can be attributed to the change in the lipid phase and to the interaction with cholesterol. It is shown that the temperature dependence of the overall bandwidth of the amide I' band displays a phase-specific behavior. In addition, it is observed that the amide I' band for a sample exhibiting phase coexistence can be described by a linear combination of the spectra of the individual lipid phases. Description of changes in the amide I' band shape and by that the study of possible hydrogen bonding interactions of sphingomyelin with cholesterol was assisted by the use of curve fitting. It turns out that the presence of hydrogen bonding between hydroxyl group of cholesterol and carbonyl group of sphingomyelin is obscured by the complexity of different possible hydrogen bonding and coupling between the N-H (N-D) and the CO group vibrations.

We used Fourier transform infrared spectromicroscopy in the attenuated total reflection configuration to study biochemical events associated with the response to light of an intact retina. We show that the technique is suitable for the detection in real time of molecular processes occurring in rod outer segments induced by light absorption. Two-dimensional correlation analysis was applied to the identification and interpretation of specific spectral changes associated to the evolution of the system. The technique allows us to observe an extensive protein translocation, which we interpret as arising from the release of transducin from the disk membrane and its redistribution from the outer segment towards the inner segment of rod cells. These results are in full agreement with our current understanding of retinal physiology and validate the technique as a useful tool for the study of complex molecular processes in intact tissue. [figure: see text] Spectral changes in the mid infrared region following exposure of an intact retina to light.

By using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and curve fitting we have examined temperature dependence and composition dependence of the shape of the carbonyl band in phosphatidylcholine/cholesterol model membranes. Membranes were hydrated either in excess water or in excess deuterated water. The studied binary mixtures exhibit different lipid phases at appropriate temperature and amount of cholesterol, among them also the so-called liquid-ordered phase. The results confirm that cholesterol has a significant indirect influence on the carbonyl band through conformational and hydration effects. This influence was interpreted in view of the known temperature composition phase diagrams for inspected binary mixtures. In addition, direct interaction was observed, which could point to the presence of hydrogen bond between cholesterol and carbonyl group. This direct interaction, though weak, might play at least a partial role in the stabilization of cholesterol-rich lipid domains in model and biological membranes.

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.

Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4). The solvent-derived coordination of the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminal water/hydroxide bound to Fe(2+) but no terminal water/hydroxide bound to Fe(3+). The terminal water is lost upon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysis mechanism involving a terminal Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Surface-enhanced resonance Raman scattering (SERRS) spectra were measured for the beta-carotene and lycopene carotenoids present in low-density lipoproteins (LDLs), which were isolated from human plasma and adsorbed on roughened silver surfaces. The silver surface was modified by formation of a self-assembled monolayer (SAM) of carboxylate-terminated linear alkanethiols in order to simulate the LDL binding region of the cellular LDL receptor. Thiols of different chain length were used to produce SAMs of varying thicknesses. It was shown that carotenoids are not released from the LDL particle upon adsorption onto the bare and thiol modified silver surfaces. The SERRS studies indicated that beta-carotene and lycopene were present in the shell of the LDL particle. The dependence of SERRS on the distance from the silver surface was different for beta-carotene and lycopene in LDL. This observation suggests that the two carotenoids are located in different places of the LDL particle.

Tyrosines can be selectively nitrated in a protein and the resultant chromophore can be used as an in situ probe of the tyrosine environment. Resonance Raman scattering could have specific advantages as a detection method because of the inherent selectivity of the technique and because shifts in the intensity and frequency of the nitro stretch can be detected and related to the form and environment of the nitrotyrosine. To evaluate this possibility the internal residue Tyr67 of cytochrome c was nitrated and resonance Raman scattering was recorded. With 413.1-nm excitation the resonance scattering from the heme protein dominates, but with 457.9-nm excitation intense bands due to nitrostretching vibrations are readily observed. The frequency of the internal Tyr67 indicates an aqueous environment that suggests that on nitration this residue becomes exposed on the protein surface or that water enters the active pocket. pH dependent measurements can be used to follow the protonation of the residue. A pK(a) of approximately 7 also indicates an aqueous environment. This initial study indicates that resonance Raman scattering does have unique advantages as an in situ probe of the local structure of nitrated tyrosine residues.

The reaction of mammalian cytochrome P-450 2B4 with nitrogen monoxide and oxygen has been studied by surface-enhanced resonance Raman scattering (SERRS) to obtain sharp and definitive information in situ on the nature of the changes in the active site pocket. The initial reaction produces a six co-ordinate low spin haem-nitrogen monoxide adduct. A slower reaction leads to the irreversible formation of a five co-ordinate high-spin iron (III) haem with no nitrogen monoxide bound to it and to the nitration of an aromatic side chain, probably a tyrosine, in the proximity of the active site. In the presence of excess nitrogen monoxide, the second reaction is controlled by oxygen concentration. The sequence of events corresponds to the biphasic inhibition induced by NO in other cytochromes P-450 and peroxidases and is postulated to occur by the formation of a nitrating agent at the haem followed by diffusion to the tyrosine. The nitrated amino acid and the oxidation and spin state of the haem are observed easily by SERRS with low concentrations of protein making it a particularly suitable method for the investigation of reactions of NO in complex biological matrices.