The nature of bile salt micelles as studied by deuterium NMR.

Deuterium NMR of 3alpha,12alpha-dihydroxy-7,7dideutero-5beta-cholanic acid was studied. Molcular sizes obtained from deuterium spin-lattice relaxation time (T1) data of 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid in methanol and in water are in accordance with monometic and tetrameric structures in the two media, respectively. The deuterium T1 and intensity of 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid in aqueous solution at pH 8.0--8.8 were studied as functions of NcC1 and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaC1, and that 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

The interaction of amino-deuteromethylated melittin with phospholipid membranes studied by deuterium NMR

Melittin, deuteromethylated on each of the four amino groups (Gly-1 N alpha and Lys-7, 21, and 23 N epsilon), was prepared by reductive methylation using deuteroformaldehyde and NaBD3CN. Deuterium NMR spectra were obtained for the modified peptide (D-melittin) bound to phospholipid bilayers and erythrocyte ghosts. D-Melittin at 4 mol% (peptide:lipid) induced reversible transitions between extended bilayers and micelles at the phase-transition temperature in dimyristoylphosphatidylcholine (DMPC) bilayers. These changes in lipid morphology did not occur at 1 mol% D-melittin: DMPC and the peptide was highly motionally restricted in gel in gel-phase lipid.     

Partitioning behaviour of 1-hexanol into lipid membranes as studied by deuterium NMR spectroscopy

Deuterium nuclear magnetic resonance (NMR) spectroscopy was used to study the partitioning behaviour of 1-hexanol specifically deuterated in the alpha-position into model lipid bilayers. In all systems studied, the observed deuterium NMR lineshapes were time-dependent. Initially, 1-hexanol-d2 gave rise to an isotropic deuterium resonance with a different chemical shift from that of aqueous 1-hexanol-d2. After equilibration over a period of days, a broader spectral component characteristic of a spherically-averaged powder-pattern was observed. The quadrupole anisotropy of the 1-hexanol-d2 giving rise to the broad spectrum depended upon the cholesterol content of the membrane. From quantitation of the anisotropic to isotropic deuterium NMR spectra, the partition coefficients of 1-hexanol-d2 in a number of bilayer systems (asolectin and phosphatidylcholine bilayers (the latter with and without cholesterol] were determined. The partitioning of 1-hexanol-d2 into red blood cell membranes, and a suspension of lipids extracted from red blood cell membranes, was also examined. It is suggested that 1-hexanol, and probably other lipophiles, can partition to either the bilayer surface or the bilayer interior in a time-dependent manner.     

Micelle formation of bile salts and zwitterionic derivative as studied by two-dimensional NMR spectroscopy

The self-association of sodium taurodeoxycholate (NaTDC) and a zwitterionic derivative of cholic acid (CHAPS) in deuterium oxide was investigated by one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts of several protons suggested that NaTDC and CHAPS form nonamers and heptamers, respectively, as well as dimer. The equilibrium constants of dimerization and the micellar aggregation numbers are close to the literature values. From the intensities of intermolecular cross-peaks in the nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectra of NaTDC and CHAPS micellar solutions, partial structures of their micelles were estimated. The CHAPS micelle consists mainly of the back-to-back association, similarly to taurocholate (NaTC). However, the NaTDC micelle consists of the back-to-face association, because the face of NaTDC is rather hydrophobic. Furthermore, the back of bile molecules forms a convex plane and the face forms a concave plane. The back-to-face structure of NaTDC will be stabilized by a close contact between these planes. The chemical shift changes of several protons of CHAPS and NaTC in the micellar state are close to each other, but are different from those of NaTDC. This finding is consistent with the difference in their micellar structures.     

Influences of membrane curvature in lipid hexagonal phases studied by deuterium NMR spectroscopy

The presence of reversed hexagonal phase, HII, favoring lipids in membranes has been proposed to be significant in various biological processes. Therefore an understanding of the HII phase and the transition from the lamellar to hexagonal phase is of importance. We have applied deuterium NMR spectroscopy to study the bilayer and reversed hexagonal phases of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamin e. The difference in packing between the HII and L alpha phases leads to smaller segmental order parameters in the former case. Since the order profiles are sensitive to the geometry of the aggregates, they can be used to extract structural information about the phases. We present a new means of calculating the radius of curvature, R1, for the HII phase from 2H NMR data. This method gives a value of R1 = 18.1 A, which is in agreement with current understanding of the structure of the HII phase and with x-ray diffraction data.     

The interaction of bile salt micelles with the dansyltyrosine derivatives of porcine colipase

The interaction of bile salt micelles with the tyrosines of pancreatic colipase was assessed by steady-state and time-resolved fluorescence techniques. Dansyltyrosine fluorescence showed that Tyr-55 was located in the proposed interface recognition site. In support of this claim was a 70 nm blue shift and 4.3-fold quantum yield increase in emission spectrum due to taurodeoxycholate (TDOC) micelle-complex formation. Complex formation also caused a shift in the center of the major lifetime distribution from 11.7 to 15.1 ns, and more than doubled the polarization and anisotropy decay parameters. These data supported an earlier model of colipase-micelle binding that suggested that Tyr-55 was inserted into the interior of the TDOC micelle upon binding (J.C. McIntyre, P. Hundley and W.D. Behnke, Biochem. J. 245 (1987) 821). Identical experiments on a DNS-Tyr-59 derivative of colipase showed that Tyr-59 did not specifically interact with micelles. Moreover, acrylamide quenching data suggest an alteration in the protein environment surrounding DNS-Tyr-59 such that during complex formation, the efficiency of quenching of DNS-Tyr-59 increases.     

Chain dynamics of selectively deuterated fatty acids in high-density lipoproteins studied by deuterium NMR

Deuterium order parameters have been determined for approximately 5 mol% selectively deuterated palmitic acid incorporated into the outer monolayer of high-density lipoproteins (HDL3). The values are SCD = 0.38 for [2,2-2H2]palmitic acid, 0.38 for [4,4-2H2]palmitic acid, 0.37 for [5,5,6,6-2H4]palmitic acid, 0.23 for [11,11,12,12-2H4]palmitic acid, and 0.05 for [16,16,16-2H3]palmitic acid. Comparison of the acyl chain order parameters in HDL3 with acyl chain order parameters determined recently [Parmar, Y.I., Wassall, S.R., & Cushley, R.J. (1984) J. Am. Chem. Soc. 106, 2434-2435] for approximately 5 mol% deuterated palmitic acid in sonicated unilamellar vesicles, composed of the same ratio of phosphatidylcholine/sphingomyelin (85/15 w/w) found in HDL3, shows that acyl chain order in the HDL3 monolayer is approximately 3-5 times higher than in the vesicle bilayer. The acyl chain order in the lipoprotein monolayer is approximately 1.5-2 times higher than in the bilayer of phosphatidylcholine multilamellar dispersions. Deuterium longitudinal relaxation times have been measured for deuterated palmitic acid in HDL3, and the values T1 approximately 16 ms for C2H2 and 170 ms for C2H3 groups are a factor of more than 2 times smaller than found in phospholipid bilayers.     

The state of water in biological systems as studied by proton and deuterium relaxation

Careful experiments on the measurement of the intensity of the deuterium NMR signal for ²H₂O in muscle and in its distillate were performed, and they showed that all ²H₂O in muscles is “NMR visible.”The spin-lattice relaxation time (T₁) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to −70°C. T₁ values of deuterons in ²H₂O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to −20°C. Calculations on T₁ for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T₁ values compared to pure water and the frequency dependence of T₁ are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

The state of water in muscle as studied by pulsed NMR

Spin-lattice relaxation times for the water protons in rat gastronemius muscle are reported over the temperature range +37 to −70°C at six resonance frequencies ranging from 4.5 to 60.0 MHz. From −8 to −70°C, the bulk of the muscle water is frozen. The unfrozen part is termed the hydrated layer and amounts to 7–12% of the total water content. Its correlation time takes teh form of a log-Gaussaian distribution function. From +37 to −8°C, the spin-lattice relaxation time is explained by the exchange of water between the hydration layer and the rest of the water, which behaves like ordinary liquid water. The fact that the observed T₂ values are smaller than the calculated values is attributed to the inner field inhomogeneity of the heterogenous system and/or the modification of T₂ due to non-zero dipolar interaction.In the presence of perdeuterated dimethylsulfoxide, the freezing point of water decreases and the amount of non-freezable water increases. T₁ of water protons for muscle containing 10, 20, and 40% dimethylsulfoxide was calculated.     

Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles.

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.     

Sterol orientations in phosphatidylcholine liposomes as determined by deuterium NMR

Deuterium magnetic resonance spectra (55.26 MHz) of cholesterol-3 alpha-d1 and epicholesterol-3 beta-d1 in dipalmitoylglycerophosphocholine (DPPC) liposomes were measured as a function of sterol-to-phospholipid ratio below (24 degrees C) and above (60 degrees C) the phase transition temperature of DPPC. From the quadrupolar splittings delta vq, the molecular order parameters S describing the motions of the sterols in the bilayer were calculated, and the most probable angle of tilt alpha 0 of the molecular axis of the sterols relative to the bilayer normal was determined. We observed that the molecular axis of cholesterol in DPPC liposomes at both 24 and 60 degrees C is tilted at an angle of 16-19 degrees with the 3 beta-hydroxyl group projecting parallel to the bilayer normal into the aqueous interface. In contrast, at 24 degrees C, epicholesterol is aligned parallel (0 degrees) to the bilayer normal, placing the 3 alpha-hydroxyl group essentially perpendicular to the bilayer normal along the aqueous interface. At 60 degrees C, the average angle of epicholesterol (16-18 degrees) is similar to that of cholesterol, which can project the 3 alpha-hydroxyl group into the hydrophobic bilayer region. On the basis of the observed tilt angles of the two isomeric sterols in DPPC liposomes, a model is proposed that can rationalize the differential effects of cholesterol and epicholesterol on membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic and molecular determinants of sterol solubilities in bile salt micelles

We examined, by reverse-phase high performance liquid chromatography (HPLC), the hydrophilic-hydrophobic balance of cholesterol and 12 non-cholesterol sterols and related this property to their equilibrium micellar solubilities in sodium taurocholate and sodium glycodeoxycholate solutions. Sterols investigated exhibited structural variations in the polar function (3 alpha-OH, 3 beta-OH, 3 beta-SH), nuclear double bonds (none, delta 5, or delta 7), side chain length (C27, C28, C29) and side chain double bonds (none, delta 22, or delta 24). In general, a sterol's hydrophilic-hydrophobic balance became progressively more hydrophobic (as exemplified by increasing HPLC retention values, k') with additions of side chain methyl (C28) and ethyl (C29) groups and with 3 beta-SH substitution of the 3-OH polar function. Side chain delta 22 and especially delta 24 double bonds rendered the sterols appreciably more hydrophilic, whereas a single nuclear double bond had little influence. Sterol solubilities (24 degrees C, 0.15 M Na+) were uniformly greater in 50 mM solutions of sodium glycodeoxycholate (range 0.15 to 2.5 mM) than in equimolar solutions of the more hydrophilic bile salt, sodium taurocholate (range 0.07 to 0.67 mM). For each bile salt system, a strong inverse correlation existed between micellar solubilities of sterols and their HPLC k' values, indicating that more hydrophilic sterols had greater micellar solubilities than the more hydrophobic ones. Based upon the aqueous monomeric solubilities of cholesterol (C27) and beta-sitosterol (C29) at 24 degrees C, we derived free energy changes associated with micellar binding and found that solubilization of both sterols was more energetically favored in glycodeoxycholate solutions. Although cholesterol exhibited a higher binding affinity than beta-sitosterol in glycodeoxycholate micelles, solubilization of beta-sitosterol in taurocholate micelles was more energetically favored than cholesterol by -0.6 kcal/mol. Based upon these results we offer a thermodynamic explanation for the greater micellar solubilities of more hydrophilic sterols and suggest that the high affinity, but low capacity, of a typical phytosterol for binding to trihydroxy bile salt micelles may provide a physical-chemical basis for its inhibition of intestinal cholesterol absorption.     

Mechanism of urocanase as studied by deuterium isotope effects and labeling patterns

Nicotinamide adenine dinucleotide (NAD) dependent urocanase (4'-imidazolone-5'-propionate hydro-lyase, EC 4.2.1.49) from Pseudomonas putida was found to catalyze an exchange reaction between solvent and the 4'-hydrogen of urocanate or imidazolepropionate at a rate faster than that of overall deuterium was compared to unlabeled urocanate as a substrate, no isotope rate effect was noted. For examination of the possibility of an NAD+-mediated intramolecular hydride transfer of the 4'-hydrogen to a position on the side chain of oxoimidazolepropionate, the origins of hydrogen at positions 2 and 3 in the propionate chain were studied as a function of reaction time and extent of exchange of the 4'-hydrogen. No transfer of hydrogen from the 4' position to the side chain was observed, thereby eliminating mechanisms requiring hydride transfer via NADH between these positions. Catalytic rates in 1H2O vs. 2H2O revealed a 3-fold difference which was ascribed to a rate-limiting proton addition step. Similarly, a 5-fold decrease in Vmax was found for the reverse reaction when oxoimidazole[2,3-2H2]propionate was compared to unlabeled oxoimidazolepropionate. These data support a mechanism involving water addition across the conjugated double bond system of urocanate, rather than an internal oxidation--reduction process, yet NAD+ is required. A mechanism is proposed which uses electron delocalization in the imidazole nucleus, via an imidazole--NAD adduct, to facilitate water attack and subsequent formation of oxoimidazolepropionate.     

The state of water in biological systems as studied by proton and deuterium relaxation.

Careful experiments on the measurement of the intensity of the deuterium NMR signal for 2-H2 O in muscle and in its distillate were performed, and they showed that all 2-H2 O muscle is "NMR visible". The spin-lattice relaxation time (T1) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to --70 degrees C. T1 values of deuterons in 2H2 O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to --20 degrees C. Calculations on T1 for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T1 values compared to pure water and the frequency dependence of T1 are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

The state of water on hydrated collagen as studied by pulsed NMR

The spin-lattice relaxation time (T₁) of water adsorbed on collagen fibers was determined at six frequencies and temperatures varying from 25° to ?80°C. Care was taken to eliminate the contributions to the signal of protons other than those in the adsorbed water. Quantitative calculations were made on T₁ and the results were compared with the experimental data. It is suggested that a maximum of about 0.50–0.55 g water per g collagen forms a hydration layer, which cannot be frozen down to ?90°C and exhibits a distribution of motional correlation times. For collagen samples containing a larger quantity of adsorbed water, the additional water molecules behave like ordinary isotropic water, having a single correlation time and a freezing temperature of about ?10°C.