Conformational features of cell-bound drugs as detected by selective proton relaxation rate investigations

The nonselective and selective longitudinal relaxation rates were measured for procaine protons in the presence of model lipid membranes, biological membranes and whole cells. Unlike the nonselective relaxation rates, the selective rate was shown to be particularly sensitive in detecting binding interactions with macromolecular cell constituents. It was shown that the aromatic moiety of procaine is involved in binding the cell plasma membrane.     

Measurement of proton relaxation rates in proteins

Summary Five different types of experiment are described which make it possible to measure various relaxation rates of selected protons in crowded spectra of macromolecules such as proteins: longitudinal spin-lattice relaxation rates =1/T₁, transverse relaxation rates =1/T₂ measured under conditions of free precession, transverse relaxation rates ¹ _LOCK=1/T₁ measured under conditions of spin-locking, and transverse relaxation rates ^DQC=1/T₂ ^DQC and ^ZQC=1/T₂ ^ZQC of double- and zero-quantum coherences. The surprisingly large discrepancy between the transverse rates ^t and ^t is discussed in detail. To separate overlapping proton signals, the experimental schemes involve one or several magnetization transfer steps, using a doubly selective homonuclear Hartmann-Hahn method. Numerous variants of the basic ideas can be conceived, depending on the extent of signal overlap and on the topology of the networks of scalar couplings. Applications are shown to H and H of Tyr²³, to H, H and H of Cys³⁰, and to H and H of Ala²⁴ in bovine pancreatic trypsin inhibitor (BPTI).     

A study of dextran hydration in dilute,aqueous solution by the proton magnetic relaxation method

The times of proton magnetic relaxation in dilute (<1%), aqueous solutions of dextrans, having a molecular weight range of 17 x 10³?500 x 10³, are highly sensitive to the temperature—time prehistory of the samples investigated. Reliable results have been obtained only after preliminary heating of the solutions at 100° for 30 min. On the basis of the model of “two states of water in a solution”, the dependence of the degree of hydration of a dextran on its molecular weight has been obtained. In the molecular weight range 17 x 10³?110 x 10³, only a fraction of the D-glucose residues are hydrated, the degree of hydration increasing with the molecular weight. The data obtained are considered to be a consequence of intersegmentary interaction in a dextran macromolecule.     

Field-dispersion profiles of the proton spin-lattice relaxation rate in chloroplast suspensions. Effect of manganese extraction by EDTA,Tris, and hydroxylamine

Proton spin-lattice relaxation rates (R₁) have been measured in a variety of dark-adapted chloroplast suspensions over a range of field strengths between 1 and 15 kG (4–5 MHz). When the effects of EDTA or Tris washing on chloroplast relaxivities are compared, the pool of Mn associated with oxygen evolution is seen not to contribute significantly to relaxivity. Instead, nearly all of the observed relaxivity, which is characterized by a paramagnetic maximum near 20.7 MHz in the field dispersion profile of R₁, appears to arise from contaminating non-functional Mn(II) that can be removed by EDTA during the isolation procedure. These observations, which contradict previous reports ascribing chloroplast relaxivity to the water-oxidizing system, require a reevaluation of proposed models, derived from NMR studies, of the state of Mn in the water-splitting reaction.Chloroplasts from which loosely bound non-functional Mn has been removed by EDTA washing do show an enhancement of relaxivity when exposed to NH₂OH at concentrations known to inactivate water oxidation. This NH₂OH-induced relaxivity is comprised of Mn(II) in two distinct paramagnetic sites. One site is chelatable by EDTA, whereas the other site is not. This finding suggests that some Mn(II) tightly bound to thylakoid membranes can contribute to relaxivity after inactivation of the oxygen-evolving reaction.     

Chlorophyll Fluorescence of Lichens Containing Green and Blue-Green Algae During Hydration by Water Vapor Uptake and by Addition of Liquid Water*

The fluorescence yield at room temperature of the lichens Ramalina maciformis and Peltigera rufescens, containing either green or blue-green algae (Cyanobacteria) as phycobionts, has been investigated during rehydration of the dry lichens by water vapor uptake or by wetting with liquid water. In the dry state the fluorescence yield with all reaction centers open, F_o, was low and no variable fluorescence could be induced with both species. Whereas R. maciformis, containing green algae, regained normal fluorescence behavior during water vapor uptake, the photosynthetic apparatus of the blue-green algae-containing P. rufescens stayed inhibited and could be reactivated only by addition of liquid water. During stepwise rehydration at increasing air humidities, a pattern was established for the recovery of the different fluorescence parameters in R. maciformis. At a dry-weight related water content between 30 and 40%, F_o rose sharply. Maximal variable fluorescence yield expressed as (F_v)_m/F_o, strongly increased in the same range of water content and remained constant above a water content of 50%. Non-photochemical fluorescence quenching, q_NP, determined at the end of a period of actinic illumination, decreased with increasing water vapor uptake. While spraying the lichen with liquid water did not induce a further decrease of q_NP, slow dehydration at lowered air humidity led to a minimal value of q_NP at a water content of 65 % indicating optimal photosynthetic rate under these conditions. These results extend the conclusions drawn from earlier gas exchange experiments that blue-green algae-containing lichens are unable to reactivate photosynthesis by water vapor uptake. During a re- and de-hydration cycle, no hysteresis in the hydration dependence of the fluorescence parameters was found. From this and the presence of a stable and low F_o value at prolonged incubation in nearly water vapor saturated air, we conclude that the reactivation of photosynthesis in blue-green algae-containing lichens is not prevented through high diffusion resistances for water.     

Frequency dependence of water proton longitudinal nuclear magnetic relaxation times in mouse tissues at 20°C

We have measured the proton longitudinal relaxation times of tissue water of healthy and tumor-bearing mice as a function of the Larmor frequency in the range 6.7 to 90 MHz. These data can be rationalized according to , where A and B are constants specific to the tissue species. We present an interpretation of this frequency dependence within the Fast Exchange Two States model. It is shown that involving a distribution of correlation times for water proton-proton interaction does not yield consistent results, whereas a physically meaningful translational diffusion model pertinent to the dipolar interaction between water protons and macromolecules protons leads to the required frequency dependence. Essentially tissues would differ by the ‘bound’ versus ‘free’ proportion, or by structural properties of cells, rather than by the time-scales governing water motion.     

Determination of liposomal encapsulation efficiency using proton NMR spectroscopy

A rapid and simple approach using 1H NMR was developed for determination of liposomal encapsulation efficiency without the need for physical separation of entrapped and non-entrapped marker. Measurements were made using a marker (homocarnosine) with a pH-sensitive 1H chemical shift in the presence of a pH gradient across the phospholipid vesicle membrane, or by addition of the chemical shift reagent, thulium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-(methylene phosphonic acid sodium salt) (TmDOTP5-). The measured encapsulation efficiencies for the liposomal dispersions prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) through extrusion using 50, 200 and 1000 nm polycarbonate membranes were found to be identical using the two different experimental approaches.     

Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.

The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.     

Conformational characterization of square planar nickel(II) tetraaza macrocyclic complexes by proton NMR. Crystal structure of [Ni(13aneN4)]ZnCl4

Conformations in solution of several diamagnetic nickel(II) complexes of macrocyclic tetraaza ligands are elucidated using proton NMR. There are six possible configurational isomers of planar [Ni(13aneN₄)]²⁺ (13aneN₄ = 1,4,7,10-tetraazacyclotridecane due to the orientation of the N---H protons above or below the plane of the macrocyle. Using NMR it is shown that in aqueous solution the [Ni(13aneN₄)]²⁺ complex has the R,S,R,S or trans-II configuration. A single-crystal X-ray study demonstrates the same configuration of the nitrogen atoms in the complex [Ni(13aneN₄)]ZnCl₄. In the case of the 14-membered ring macrocyle cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane), previous NMR studies revealed the presence, in aqueous solution, of the previously unobserved trans-I or R,S,R,S isomer, whose spectrum is examined in greater detail here. Solution structures of nickel(II) complexes of bicyclam (1,5,8,12-tetraazabicyclo[10.2.2]hexadecane) and dachden (N, N′-bis(2-aminoethyl)-1,4-diazacycloheptane) are also reported.     

Mechanism of proton transport by plant plasma membrane proton ATPases

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E₁ proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

A proton nuclear magnetic resonance study on the solution structure of crotamine

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996     

The active site of Manganese-containing superoxide dismutase fromBacillus stearothermophilus studied by1H and19F magnetic relaxation

The dependence of the magnetic relaxation rates of¹H and¹⁹F on temperature, frequency, pH and N ₃ ^- concentration, were measured in solutions of Manganese-containing superoxide dismutase ofBacillus stearothermophilus, and were compared to activity measurements, in order to obtain some information on the structure and dynamics at Mn(III) present in the active site of the enzyme. The experimental data lead us to hypothesize the presence of two binding sites in the coordination sphere of the enzyme bound Mn(III), which are accessible to water and anions and have different chemical and spectroscopic properties. NMR measurements carried out in the presence of competitive inhibitors and the pH dependence of both NMR relaxation rates suggest that F^-, N ₃ ^- and OH^- ions bind to one site, while a water molecule binds to the other one. The stability constant values of the complexes between these anions and the enzyme are reported. The influence of the anions on activity and the pH dependence of NMR parameters are discussed.Abbreviations MnSOD Manganese containing superoxide dismutase     

Efficiency of proton extrusion by chemically modified mitochondria

Bovine heart mitochondria were treated with limited amounts of iodoacetamide, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, phenylglyoxal, tetranitromethane and 1-fluoro-2,4-dinitrobenzene, respectively. Examination of the respiration and proton extrusion characteristics of the chemically modified mitochondria suggests that sulfhydryl and imidazole groups are not directly involved in proton pumping, but that some of the labeled carboxyl, amino, guanidinium and phenolic groups may participate in an indirect proton-extrusion process. Cross-linking mitochondria with glutaraldehyde drastically decreases the efficiency of proton extrusion, whereas treatment of mitochondria with valeraldehyde under similar conditions did not affect the proton-pumping efficiency significantly. The latter observations show that conformational change in the inner mitochondrial membrane may play a crucial role in the active translocation of protons coupled to electron transport. Comparison of the reactivities of the essential amino and carboxyl groups in mitochondria in different oxidation states suggests that these two types of essential functional groups are more exposed to water in the oxidized state. An indirect mechanism for proton pumping based on protein conformational change driven by electron transport based on the results of the present chemical modification studies is suggested.     

Different types of biological proton transfer reactions studied by quantum chemical methods

Different types of proton transfer occurring in biological systems are described with examples mainly from ribonucleotide reductase (RNR) and cytochrome c oxidase (CcO). Focus is put on situations where electron and proton transfer are rather strongly coupled. In the long range radical transfer in RNR, it is shown that the presence of hydrogen atom transfer (HAT) is the most logical explanation for the experimental observations. In another example from RNR, it is shown that a transition state for concerted motion of both proton and electron can be found even if the donors are separated by a quite long distance. In CcO, the essential proton transfer for the OO bond cleavage, and the most recent modelings of proton translocation are described, indicating a few remaining major problems.     

Use of (1)H cross-relaxation nuclear magnetic resonance spectroscopy to probe the changes in bread and its components during aging

(1)H nuclear magnetic cross-relaxation spectroscopy was used to probe the molecular mobility/rigidity in bread and its components during storage. The Z-spectra lineshapes, attributed to the solid-like polymer fractions of the samples, differed for the bread, gelatinized waxy starch (GX), gelatinized wheat starch (GW), heated flour (HF), and heated gluten (HG). Upon storage, no change was observed in the Z-spectrum of the bread sample, while the Z-spectra for GX, GW, and HG increased in the width at half height of the decomposed broad component (increased rigidity). These trends in the Z-spectra detected by NMR were contradictory to the DSC results that showed an increase in amylopectin retrogradation enthalpy for all samples containing starch, including bread. These trends in the Z-spectra detected by NMR were not reflected by the DSC results that showed an increase in amylopectin retrogradation enthalpy for all samples, including bread. The differences in molecular mobility could not be therefore, due to recrystallized amylopectin and may be attributed to the role of gluten and/or redistribution of water in the amorphous regions of the samples.