Biomedical subjects

R Mendelsohn

Publications and source records attributed to R Mendelsohn.

At least 55 records · Page 3Linked to original sources

PubMed1996

Fourier transform infrared spectroscopy of the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite: new correlations between X-ray diffraction and infrared data.

Fourier Transform infrared spectroscopic analysis of maturing, poorly crystalline hydroxyapatite (HA) formed from the conversion of amorphous calcium phosphate (ACP) at constant pH or variable pH show only subtle changes in the v1, v3 phosphate absorption region (900 cm-1-1200 cm-1). This region is of interest because it can be detected by analysis of mineralized tissue sections using FT-IR microscopy. To evaluate the subtle spectral changes occurring during the maturation, second derivatives of the spectra were calculated. HA formed at constant pH showed little or no variation in the second derivative peak positions with bands occurring at 960 cm-1, 985 cm-1, 1030 cm-1, 1055 cm-1, 1075 cm-1, 1096 cm-1, 1116 cm-1, and 1145 cm-1. These bands can be assigned to molecular vibrations of the phosphate (PO4(3-)) moiety in an apatitic/stoichiometric environment of HA. In contrast, during the early stages of maturation of the HA formed at variable pH, second derivative peak positions occurring at 958 cm-1, 985 cm-1, 1020 cm-1, 1038 cm-1, 1112 cm-1, and 1127 cm-1 shifted in position with maturation, indicating that the environment of the phosphate species is changing as the crystals mature. Peaks at 1020 cm-1, 1038 cm-1, 1112 cm-1, and 1127 cm-1 were attributable to nonstoichiometry and/or the presence of acid phosphate-containing species. This concept was supported by the lower Ca:P molar ratios measured by chemical analysis of the synthetic material made at variable pH. Using the second derivative peak positions as initial input parameters, the v1, v3 phosphate region of the synthetic HAs prepared at constant pH were curve fit. X-ray diffraction patterns of these same materials were also curve fit to calculate the changes in crystallinty (size/perfection) in the c-axis 002 reflection as well as the 102, 210, 211, 112, 300, 202, and 301 planes. Linear regression analysis showed that the changes in the percent area of the underlying bands at 982 cm-1, 999 cm-1, 1030 cm-1, 1075 cm-1, 1096 cm-1, 1116 cm-1, and 1145 cm-1 were correlated with changes in crystallinity in one or more of the reflection planes. It is suggested that a combination of second-derivative and curve-fitting analysis of the v1, v3 phosphate contour allows the most reproducible evaluation of these spectra.

Calcium Phosphates↗

PubMed1996

Fourier transform infrared microscopy of calcified turkey leg tendon.

Fourier transform infrared microscopy (FT-IRMS) was used to monitor spatial variations in the quality and quantity of the mineral phase in calcified turkey tendon. Spectral maps were generated by analysis of 50 microns x 50 microns areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and mineralized matrix begins, revealed marked changes in the spectrally determined mineral-to-matrix ratio, whereas regions deeper into the mineralization front showed a relatively constant ratio. Since spectra of EDTA-demineralized matrix were similar to those of nonmineralized matrix, the nonmineralized regions of the tendon were used for spectral subtraction. The broad, relatively featureless contour of the mineral v1, v3 phosphate region (900-1200 cm-1) showed only subtle changes at different stages of mineralization. Second derivatives of these spectra were calculated and compared with those of synthetic, poorly crystalline hydroxyapatite (HA). The peak positions seen in second-derivative spectra of the mineral near the transitional region were within +/- 2 cm-1 of the least mature synthetic HAs whereas spectra of the mineral deeper into the mineralization front were within +/- 2 cm-1 of the most mature synthetic HAs. Spectra from cross- and longitudinal sections at equivalent positions in the tendon, and polarized FT-IRMS data were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and longitudinal sections of the tendon showed no major differences in either the v1, v3 phosphate region or the amide I, II, or III components (1200-1800 cm-1). However, polarized FT-IR spectra revealed dramatic differences in both of these regions. Despite these differences, second-derivative analysis of the v1, v3 regions revealed no significant changes in the positions of the underlying bands used to characterize the environments of the phosphate ion in poorly crystalline HA. The results of this study demonstrate the power of FT-IRMS to monitor spatial variations of the mineral phase in calcified tissue. Also, the incorporation of polarized radiation provides a method capable of assessing the molecular orientation of the mineral phase relative to the collagen matrix.

Animals↗

PubMed1996

Fourier transform infrared microspectroscopic analysis identifies alterations in mineral properties in bones from mice transgenic for type X collagen.

Type X collagen has been implicated in the morphogenetic events of endochondral ossification (EO), including the calcification of hypertrophic cartilage and trabeculae prior to their replacement by bone and marrow. Recently, transgenic mice, which expressed a truncated collagen X protein, were reported to exhibit morphologic alterations in all tissues arising through EO. Specifically, the growth plates were compressed within the zone of cartilage hypertrophy, and the number and size of calcified trabeculae were reduced. The condition in the mouse is comparable to Schmid metaphyseal chondrodysplasia in humans for which, to date, 20 defined type X collagen mutations have been reported. The transgenic mouse showed no alterations in mineralization by conventional histology, however, it did show a decrease in newly formed bony trabeculae, and a thinning of periosteal bones. Fourier transform infrared (FTIR) spectroscopy has previously been shown to provide quantitative and qualitative information about the relative amount of mineral and carbonate present, mineral composition, and crystal perfection. To determine whether the expression of abnormal collagen X molecules had an effect on mineral properties, the "quality" of mineral crystals was analyzed in thin sections of tibia from day 17 and day 25 genotypically negative (normal) and positive (mutant) mice from several independent transgenic mouse lines showing varying degrees of the mutant phenotype, by means of Fourier transform infrared microscopic analysis (FTIRM). The results indicate definite differences between normal and transgenic mice calcified cartilage mineral, both in the amount present and the "quality" of the crystals. Calcified cartilage mineral from transgenic mice exhibited less crystallinity and higher acidic phosphate content than the corresponding mineral from normal specimens.

Animals↗

PubMed1996

IR spectroscopic study of the structure and phase behavior of long-chain diacylphosphatidylcholines in the gel state.

Fully hydrated dispersions of simple linear saturated diacylphosphatidylcholines with even-numbered acyl chains of lengths from 18 to 24 carbons can exist in a low-temperature, highly ordered, orthorhombic phase (G(o)) that differs from the L beta phase (Gd) normally found for shorter chains. The temperature behavior of these dispersions has been studied by infrared spectroscopy. Chain packing in the G(o) phase was found to be nearly identical to that of the orthorhombic phase of crystalline n-alkanes. With increasing temperature, the G(o) phase undergoes a transition to Gd at approximately 45 degrees C below Tm. This transition occurs at a higher temperature and becomes sharper with increasing chain length. Chain packing in the Gd phase was found to be disordered in a way that can be expressed in terms of a distribution of subcell setting angles. The Gd phase converts to a phase (Gh) with hexagonal-like chain packing at temperatures below Tm. The results support and extend those of a recent x-ray diffraction study of the 24-carbon diacyclphosphatidylcholine gel.

Calorimetry, Differential Scanning↗

PubMed1996

Infrared reflection-absorption of melittin interaction with phospholipid monolayers at the air/water interface.

The interaction of melittin with monolayers of 1,2-dipalmitoylphosphatidylcholine and 1,2-dipalmitoylphosphatidylserine has been investigated with infrared external reflection-absorption spectroscopy. Improved instrumentation permits determination of acyl chain conformation and peptide secondary structure in situ at the air/water interface. The IR frequency of the 1,2-dipalmitoylphosphatidylcholine antisymmetric acyl chain CH2 stretching vibration decreases by 1.3 cm-1 upon melittin insertion, consistent with acyl chain ordering, whereas the same vibrational mode increases by 0.5 cm-1 upon peptide interaction with the 1,2-dipalmitoylphosphatidylserine monolayer, indicative of chain disordering. Thus the peptide interacts quite differently with zwitterionic compared with negatively charged monolayer surfaces. Melittin in the monolayer adopted a secondary structure with an amide l(l') frequency (1635 cm-1) dramatically different from the alpha-helical motif (amide l frequency 1656 cm-1 in a dry or H2O hydrated environment, amide l' frequency 1645 cm-1 in an H-->D exchanged alpha-helix) assumed in bilayer or multibilayer environments. This work represents the first direct in situ spectroscopic indication that peptide secondary structure in lipid monolayers may differ from that in bilayers.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Sexual behaviour in travellers abroad attending an inner-city genitourinary medicine clinic.

OBJECTIVE: To investigate frequency of sexual encounters with new partners abroad in patients attending a genitourinary clinic (GUM). METHODS: In a case series 464 attenders at a genitourinary medicine clinic completed an anonymous self-administered questionnaire, if they had been abroad recently, enquiring about sexual behaviour abroad. RESULTS: 28.4% of subjects admitted to sex with a new partner abroad with only 41.7 per cent consistently using condoms. There were no significant differences in condom use for gender, ethnicity or type of visit and relationship. Twenty-nine per cent of those admitting to sex abroad had more than one partner. The risk of multiple partners was not associated with gender, ethnicity, type of visit (holiday or business) or type of relationship (heterosexual or homosexual). The first partner abroad for 63% of men and 62.5% of women was of a nationality other than that of United Kingdom residents. Non-Caucasians and homosexuals were significantly more likely to have first partners abroad from outside the UK than Caucasians and heterosexuals respectively. CONCLUSION: The occurrence of casual sex abroad in GUM attenders suggests that further research is needed to establish targetable risk factors for this type of behaviour amendable to change through health promotion.

Adolescent↗

PubMed1996

Polarized FT-IR microscopy of calcified turkey leg tendon.

Polarized Fourier transform infrared microscopy (FT-IRM) was used to assess the orientation of mineral and matrix components of the normally calcified turkey leg tendon. Two groups of tendon, < 16 weeks of age (young) and > 60 weeks of age (old), were analyzed. Linear sequences from calcified, non-calcified, and transitional regions of the tendons were examined. Spectra collected in the "parallel polarization" mode were acquired with the electric vector of the infrared radiation parallel to the collagen fiber axis whereas spectra collected in the "perpendicular polarization" mode were acquired with the electric vector of the infrared radiation perpendicular to this axis. The v2 carbonate (850-890 cm-1) and v1, v3 phosphate (900-1180 cm-1) contours of the tendon mineral as well as the collagen amide I, II, and III bands of the extracellular matrix all displayed marked dichroism. The CO3(2-) ions substituted for PO4(3-) (878 cm-1, type B substitution) in the tendon mineral displayed parallel dichroism while the CO3(2-) ions substituted for OH (871 cm-1, type A substitution) in the tendon mineral displayed perpendicular dichroism. These orientational effects for both sites of carbonate substitution were greater in the older animals. The polarization properties of the v1, v3 phosphate contour were analyzed by use of an empirical anisotropy parameter (A), the value of which monitors the degree of orientation. This index significantly increased in the older animals indicating that aging produces a more highly oriented mineral. The amide I, II, and III contours of the collagen extracellular matrix also exhibited marked dichroism. The amide I component exhibits perpendicular dichroism while the amide II and III components exhibit parallel dichroism. The current study demonstrates the ability of polarized FT-IRM to assess the orientation of the mineral and matrix components of calcified tissue at the microscopic level.

Amides↗

PubMed1995

IR spectroscopic determination of gel state miscibility in long-chain phosphatidylcholine mixtures.

We report on the gel-state microaggregation in binary mixtures of diacylphosphatidylcholines over temperatures ranging from -19 degrees C to near the gel-to-liquid crystal transition. Microaggregates with lateral dimensions in the range 1-100 chains were detected and measured with an isotope infrared method that relates the splitting or the shape of the methylene scissors band to aggregate size. Measurements were made on fully hydrated dispersions of diC18DPC/diC20HPC, diC18DPC/diC22HPC, and diC18DPC/diC24HPC at molar ratios of 4:1. Low levels of aggregation were determined with reference to the spectrum of the random mixture diC18DPC/diC18HPC. For diC18DPC/diC20HPC at -19 degrees C, which previous calorimetric measurements have indicated is a nearly ideal, we found about 4% of the minority component chains to be involved in aggregates. For diC18DPC/diC22HPC, the value increased to about 11%. DiC18DPC/diC24HPC was found to be highly fractionated, in agreement with the earlier studies. The unit subcell, which defines the type of acyl-chain packing, was determined for the components of the mixtures. The temperature behavior of the phases and the temperatures at which the minority component domains undergo dissolution were determined.

Biophysical Phenomena↗

PubMed1995

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

A direct test of the "squeeze-out" hypothesis of lung surfactant function. External reflection FT-IR at the air/water interface.

The current theory of pulmonary surfactant function requires that very low surface tension be achieved and maintained in the alveolar surface film during compression (expiration). To effect this condition, it has been hypothesized that the unsaturated and/or fluid components of surfactant are selectively excluded or "squeezed out" from mixed monolayers containing both saturated and unsaturated phospholipids, leaving a surface film of essentially pure 1,2-dipalmitoylphosphatidylcholine (DPPC). External reflection Fourier transform infrared (FT-IR) spectroscopy has been employed to quantitatively test this hypothesis. Mixed monolayer films of acyl chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62) with 1,2-dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl, 2-oleoylPG (POPG), 1,2-dipalmitoylPG (DPPG) were examined in situ at the air/water interface as a function of surface pressure. The relative intensities of CD2 (CH2) stretching vibrations of the deuterated (proteated) components permitted quantitative determination of the relative concentrations of each in the film. For 7:1 (mol:mol) mixtures of DPPC-d62/DOPG, progressive, selective squeeze out of up to about 90% of the PG component is observed over a range of surface pressures from about 51 to 68 mN/m. The extent of maximal PG squeeze out was reduced to 61% for a 7:1 (mol:mol) mixture of DPPC-d62/POPG. This phenomenon, which is at least partially reversible, appears to require relatively high rates of film compression. Squeeze out was reduced (< 20%) for 7:1 (mol:mol) mixtures of DPPC-d62/DPPG or for 7:3 mixtures of DPPC-d62/POPG. Squeeze out requires that the lipid mixture achieve surface pressures greater than about 50-60 mN/m along with unsaturation (or at least conformational disorder) in the acyl chains of the non-DPPC component.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

In situ modification of the phospholipid environment of native rabbit sarcoplasmic reticulum membranes.

A water soluble hydrogenation catalyst (palladium di(sodium alizarine monosulphonate)) in a deuterium-containing environment has been used for the in situ insertion of deuterium atoms into the fatty acyl chains of biological membranes. The thermotropic response of the stretching vibrations of the formed C-D bonds, as detected by Fourier transform IR spectroscopy, was used as a selective probe of biological membrane structure. Partial deuteration of unsaturated fatty acyl chains coupled with IR detection potentially provides a means for detecting specific biological roles of particular lipid classes. In the current study of sarcoplasmic reticulum membranes and purified phospholipid/CaATPase vesicles, it is also shown that vC-D monitors change at particular membrane locations which may remain undetected through the CH2 symmetric stretching frequency, a widely used IR spectral parameter. The latter reflects the average environment of the acyl chains. The approach described here may be suitable for wide applications to the study of biomembranes.

Animals↗

PubMed1994

Adaptation to altered growth temperatures in Acholeplasma laidlawii B: Fourier transform infrared studies of acyl chain conformational order in live cells.

Acholeplasma laidlawii B cells, highly enriched in saturated C14 and C15 fatty acids, have been grown at several temperatures. Conformational order in the acyl chains of live cell membranes has been monitored with Fourier-transform infrared (FT-IR) spectroscopy. Coupled CH2 wagging progressions, characteristic of the all-trans conformation, have been used to quantitatively monitor the extent of trans-gauche isomerization. A simple model relating the changes in the progression intensity to the introduction of gauche rotamers into the acyl chains suggest that at the growth temperature (25 or 37 degrees C for the C14-enriched cells, 30 or 37 degrees C for the C15-enriched cells) the cell membrane contains about 1.5 gauche bonds per acyl chain. The thermotropic response of the CH2 stretching frequencies near 2850 cm-1 reveals that the gel-liquid-crystal phase transition is shifted toward lower temperatures when the growth temperature is reduced. In addition, at any given temperature, the cells grown at lower temperature are more conformationally disordered than their counterparts grown at higher temperature. This behavior is consistent with the quantitative results from the CH2 wagging measurements. The data reveal that A. laidlawii B cells can control the overall conformational state of their membranes and that the observed degree of disorder (1.5 +/- 0.3 gauche bonds/acyl chain), when achievable, provides optimum cell viability. The ability of this microorganism to control the degree of membrane disorder delineates one possible pathway for homeoviscous adaptation.

Acholeplasma laidlawii↗

PubMed1994

Erythrocyte peroxidation: quantitation by Fourier transform infrared spectroscopy.

We used Fourier transform infrared spectroscopy (FT-IR) to quantitatively monitor peroxidative damage in model phospholipids and in human erythrocyte membranes. The 3012-cm-1 band arising from olefinic C-H stretching modes diminished in intensity during membrane peroxidation as a result of loss of unsaturation in the phospholipid acyl chains. A precise (correlation coefficient = 0.99) linear relationship between the number of C = C bonds in the chains and the fraction of the total C-H stretching contour occupied by the olefinic C-H stretch was established for model phospholipids. Intact human erythrocytes and isolated ("ghost") membranes were also peroxidized and the loss of C = C bonds was demonstrated by FT-IR. GC studies of erythrocyte membranes confirmed that the primary site of peroxidation occurred at phospholipids containing arachidonate acyl chains. Finally, the formation of a new feature at 1260 cm-1 in the FT-IR spectrum was observed in both model compounds and erythrocyte membranes undergoing peroxidative damage. The feature corresponds to a C-O stretching and/or O-H in-plane bending vibration and is suggestive of the formation of these functional groups during peroxidation. FT-IR provides an accurate means for monitoring the effects of peroxidative damage in human erythrocytes.

Erythrocyte Membrane↗

PubMed1994

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange.

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.

Air↗

PubMed1993

Quantitative IR studies of acyl chain conformational order in fatty acid homogeneous membranes of live cells of Acholeplasma laidlawii B.

Acholeplasma laidlawii B has been grown highly enriched in myristic, pentadecanoic, and palmitic acids. The conformational order in the acyl chains of living cell membranes has been compared with that in the membranes of lysed cells and the lipids extracted therefrom, using Fourier-transform infrared (FT-IR) spectroscopic techniques. A gel-liquid-crystal-phase transition (25-45 degrees C) was noted for both the live cells and the membranes. Surprisingly, CH2 wagging progressions, characteristic of coupled oscillators from acyl chains in the all-trans conformation, were detected in the live cells and in the cell membrane. A simple model suggested the presence of 1.7 gauche bonds/chain at the growth temperature (37 degrees C) in each case. Conformational order in the live cells and in the membranes was virtually identical over the range of cell viability (5-40 degrees C), as measured by the thermotropic responses of the k = 1 and k = 2 components of the wagging mode progression. This result was confirmed by studies of the thermotropic response of the symmetric CH2 stretching vibrations, a qualitative index of acyl chain order. In contrast, the membrane lipid extracts (i) show much more conformational disorder from 5 to 25 degrees C than either the live cells or the membranes, (ii) undergo a gel-liquid transition over a broader temperature range and with a reduced magnitude of change in the symmetric CH2 stretching frequency, and (iii) demonstrate a second transition centered at 50 degrees C, which is detected by a large increase in the localized CH2 wagging mode (1368 cm-1) that arises from conformationally disordered kink+gtg states.(ABSTRACT TRUNCATED AT 250 WORDS)

Acholeplasma laidlawii↗

PubMed1993

Infrared spectroscopic study of a "hard-to-exchange" phospholipid population in the vicinity of CaATPase.

CaATPase from rabbit sarcoplasmic reticulum (SR) has been isolated, purified, and reconstituted into unilamellar vesicles with 1,2-dipentadecanoylphosphatidylcholine (DPePC) and acyl-chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62). For total lipid:protein mole ratios between 25 and 70, a constant "hard-to-exchange" phospholipid population (HEPP) of 12 +/- 4 native phospholipid molecules per protein monomer is observed, consistent with the studies of Bick et al. [(1991) Arch. Biochem. Biophs. 286, 346-352]. Thermotropic and conformational properties of the lipids in native SR and in the reconstituted systems were probed with FT-IR spectroscopy. The native SR phospholipids undergo a broad phase transition centered at about 1-2 degrees C and are thus disordered under physiological conditions. The thermotropic behavior of CH2 wagging progressions characteristic of the palmitate chains differs from that of the total lipid population and is suggestive of membrane microheterogeneity. The individual thermotropic and conformational properties of the HEPP and the exogenous lipid in reconstituted vesicles containing CaATPase and DPPC-d62 were monitored. At temperatures below the onset of the gel-liquid-crystal phase transition, the HEPP possesses significant conformational disorder, and exhibits the monotonic introduction of gauche rotamers as the temperature is raised from -55 to 27 degrees C, in contrast to the exogenous lipid, which exhibits a constant high order over the same temperature range. Nevertheless, the HEPP undergoes a residual order-disorder phase transition with similar but not identical parameters (half-width, midpoint temperature) to the gel-liquid-crystal transition of the bulk (exogenous) lipid in the reconstituted systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Labeling of tyrosines in proteins with [15N]tetranitromethane, a new NMR reporter for nitrotyrosines.

Lysozyme and ribonuclease were used as model proteins to explore the feasibility of detecting protein-bound nitrotyrosines by 15N-NMR spectroscopy. The reporter group was introduced via synthesized [15N]tetranitromethane. Several experiments for detection of the 15N resonance in the model [3-15N]nitrotyrosine demonstrated a substantial pH-dependence of the chemical shift. When lysozyme was nitrated, either two or three 15N resonances were detected, depending on the extent of nitration. The pH-dependence of the detected resonances clearly described an apparent microscopic pK in accord with reported values, while addition of Gd(III) gave selective line broadening, indicating that the 15N reporter group could also monitor relative distances from paramagnetic sources. Nitration of ribonuclease showed five 15N resonances, of which three persisted in the purified monomer. The pH-dependence of these resonances also described apparent microscopic pK values. The [3-15N]nitrotyrosine model was reduced to the [3-15N]aminotyrosine and its 15N resonance was easily monitored by several methods, including selective population inversion. When the protein-bound nitrotyrosines were similarly reduced, much sample decomposition resulted, a possible result of photooxidation, and/or reduction of disulfide bond(s), thereby making interpretation difficult.

Animals↗

PubMed1993

Calcium ion interactions with insoluble phospholipid monolayer films at the A/W interface. External reflection-absorption IR studies.

External reflection Fourier transform infrared (FT-IR) experiments are reported for insoluble monomolecular films of an equimolar mixture of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylserine (DPPS) at the A/W interface as a function of surface pressure and Ca2+ ion presence. The separate components showed a surface pressure-induced conformational ordering of the acyl chains. The conformational ordering occurred more cooperatively for the DPPS. Acyl chain perdeuteration of the DPPC permitted the observation of the response of the individual components in the binary mixture to changes in surface tension and to the presence of Ca2+. Plots of surface pressure versus CH2 or CD2 stretching frequencies were analyzed with a two-state model. At each surface pressure within the two-state region, the fraction of disordered form was the same for each lipid component, suggesting that they are well mixed on the surface. Calcium ion (5 mM in the subphase) produces almost no effect on the pressure-induced acyl chain ordering of the DPPC in a single component film, whereas the same levels of Ca2+ induce acyl chain ordering at all surface pressures in both components of the binary mixture. Thus, unlike the bulk phase mixture of DPPC/DPPS, the binary lipids in this mixed monolayer film appear to retain their miscibility in the presence of Ca2+. Finally, Ca(2+)-induced dehydration of the phosphate group was observed through characteristic frequency shifts in the asymmetric PO2- stretching mode.

1,2-Dipalmitoylphosphatidylcholine↗