NBD Group Research Articles

Interfacial Activation of Porcine Pancreatic Phospholipase A2 Studied with 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Lipids

The interfacial activation of porcine pancreatic phospholipase A2 (PLA2) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C12-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C12-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C6-NBD-PC, detect latency times of PLA2 action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C12-NBD-PC, a substrate analog, but also of product derivative C12-NBD-FA. Ultrafiltration experiments show that C12-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C12-NBD-PC fluorescence was insensitive to products, whereas C12-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C12-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C12-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C12-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield.

Predictive inferencing in adults with right hemisphere brain damage.

Predictive inferencing was evaluated in 13 adults with right hemisphere damage (RHD) and 11 adults without brain damage (NBD). Brief narrative stimuli that strongly suggested a single outcome were constructed to vary recency of mention of inference-related information. Reading times were recorded for narrative-final sentences that disconfirmed the target inferences. Slowed reading time on the final sentences was an indicator of inference generation. Adults with RHD generated target predictive inferences in contexts with recent mention of strongly biasing inference-related information. This group also evidenced maintenance of inferences over time, but to a lesser degree than participants in the NBD group. Overall, individuals with better auditory comprehension or larger estimated working memory capacity tended to maintain inferences better than did the other participants. The results are discussed in relation to current hypotheses of inferencing and discourse comprehension in adults with RHD.

Direct observation of the target cell for leaf-movement factor using novel fluorescence-labeled probe compounds: fluorescence studies of nyctinasty in legumes. Part 1

We synthesized fluorescence-labeled probe compounds bearing AMCA ( 1 ), NBD ( 2 ), and dansyl ( 3 ) groups as the fluorescent functionality. In these probe compounds, NBD-type probe, 2 , showed leaf-opening activity at 5×10 −6 M. The bioactivity of 2 is one-fifth as strong as that of the natural product, potassium isolespedezate ( 6 ). We carried out the binding experiment using 1 in a plant body.

Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter.

The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degrees C was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.

Dynamics of Membrane Penetration of the Fluorescent 7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl (NBD) Group Attached to an Acyl Chain of Phosphatidylcholine

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.

Probing the binding domain of the Saccharomyces cerevisiae alpha-mating factor receptor with rluorescent ligands.

Three analogues of the alpha-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity. The fluorescence emission maximum of the NBD group in [K7(NBD),Nle(12)]-alpha-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor. Fluorescence quenching experiments revealed that the NBD group in [K7(NBD),Nle(12)]-alpha-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer. In contrast, the emission maximum of NBD in [K7(ahNBD),Nle(12)]-alpha-factor or [Orn7(NBD),Nle(12)]-alpha-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor. The fluorescence investigation suggests that when the alpha-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.

Transmembrane movement of diether phospholipids in human erythrocytes and human fibroblasts.

We have synthesized spin-labeled (SL) and fluorescently labeled diacyl, 1-alkyl-2-acyl-, and di-alkyl glycerophospholipids. The sn-2 chain was a short chain with either a nitroxide group or a 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD). After incorporation in the exoplasmic leaflet of human erythrocytes, we found that SL-phosphatidylcholine (PC) redistributed very slowly across the plasma membrane, less than 20% reaching the cytoplasmic leaflet in 3 h at 37 degrees C. In contrast, SL-phosphatidylserine (PS) accumulated on the cytoplasmic leaflet with the same plateau corresponding to 90% of the probes inside. The characteristic times for inward redistribution were different for the three PS analogues: at 37 degrees C, the t(1/2) for the diacyl, alkyl-acyl, and dialkyl compounds were 2.3, 3.5, and 41 min, respectively. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid translocation of the PS derivatives. The diether PS bearing an NBD group translocated very slowly in human erythrocytes and no acceleration by ATP could be measured. On the other hand, in human fibroblasts, the diether NBD-PS and SL-PS were both transported from the exoplasmic to the cytoplasmic monolayer of the plasma membrane as it is the case for the transport of the respective diester PS analogues. These results prove that the ether bonds do not prevent completely PS binding and translocation by the aminophospholipid translocase despite a probable hindrance due to the ether linkage on the sn-2 chain. Because of the high stability of the ether linkage, SL and NBD diether analogues should be useful to investigate lipid traffic in cultured cells.

NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.

Cholesterol Distribution in Living Cells: Fluorescence Imaging Using Dehydroergosterol as a Fluorescent Cholesterol Analog

Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis.

Reliability and validity of an auditory working memory measure: Data from elderly and right-hemisphere damaged adults

The use of non-standardized measures in research and clinical assessments creates difficulties with interpretation and generalization of results obtained. One example of a widely used non-standardized tool is the reading/listening span paradigm for assessment of working memory (WM). WM is an important construct because of its purported relationship to language comprehension and capacity theories of cognition. This paper investigates several facets of reliability and validity for an auditory working memory measure designed for older adults and individuals with right hemisphere brain damage (RHD). Results from 28 non-brain-damaged subjects (NBD) and 11 RHD subjects indicate that the measure is internally consistent and reliable over time. Construct validity evidence, which compares favourably with evidence from existing literature, suggests that for NBD sujects this tool differentiates WM from simple short term memory. RHD subjects do not demonstrate the same pattern of validity results as the NBD group. Further evaluation with RHD patients is warranted, because clinically this tool may be useful as a measure of severity or a prognostic indicator of language comprehension abilities for this population.

Micellar Organization and Dynamics: A Wavelength-Selective Fluorescence Approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitor directly the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. We have previously shown that REES and related techniques (wavelength-selective fluorescence approach) offer a novel way to monitor organization and dynamics of membrane-bound probes and peptides. In this paper, we report REES of NBD-PE, a phospholipid whose headgroup is covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety, when incorporated into micelles formed by a variety of detergents (SDS, Triton X-100, CTAB and CHAPS) which differ in their charge and organization. In addition, fluorescence polarization of NBD-PE in these micelles shows both excitation and emission wavelength dependence. The lifetime of NBD-PE was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD group in micellar environments. Taken together, these observations are indicative of the motional restriction experienced by the fluorophore when bound to micelles. Wavelength-selective fluorescence promises to be a powerful tool for studying micellar organization and dynamics.

7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior

Steady-state and time-resolved fluorescence properties of the 7-nitrobenz-2-oxa-1, 3-diazole-4-yl (NBD) fluorophore attached either to the sn-2 acyl chain of various phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid) or to the polar headgroup of phosphatidylethanolamine were studied after insertion of these NBD-labeled lipid probes into unilamellar vesicles of phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine. The fluorescence response of the NBD group was observed to strongly depend on the chemical structure and physical state of the host phospholipids and on the chemical structure of the lipid probe itself. Among the various fluorescence parameters studied, i.e., Stokes' shifts, lifetimes, and quantum yields, the quantum yields were by far the most affected by these structural and environmental factors, whereas the Stokes' shifts were practically unaffected. Thus, depending on the phospholipid probe and the host phospholipid, the fluorescence emission of the NBD group was found to vary by a factor of up to 5. Careful analysis of the data shows that for the various couples of probe and host lipid molecules studied, deexcitation of the fluorophore was dominated by nonradiative deactivation processes. This great sensitivity of the NBD group to environmental factors originates from its well-known solvatochromic properties, and comparison of these knr values with those obtained for n-propylamino-NBD in a set of organic solvents covering a large scale of polarity indicates that in phospholipids, the NBD fluorophore experiences a dielectric constant of around 27-41, corresponding to a medium of relatively high polarity. From these epsilon values and on the basis of models of the dielectric transition that characterizes any water-phospholipid interface, it can be inferred that for all of the phospholipid probes and host phospholipids tested, the NBD group is located in the region of the polar headgroups, near the phosphoglycerol moiety of the lipids.

Photoisomerization of norbornadiene group in a rigid bichromophoric compound initiated by remote keto chromophore: long‐distance through‐bond triplet energy transfer

AbstractA diad compound 3β(bicyclo[2,2,1]hepta‐2,5‐diene‐2‐methylcarboxylate‐3‐carboxy)‐an‐drost‐5‐en‐17‐one (NBD‐S‐ONE) was synthesized and its photochemistry was examined. Irradiation of NBD‐S‐ONE in acetonitrile at λ > 300 nm selectively excited the keto chromophore. After intersystem crossing, the triplet energy of the keto group was transferred to the NBD group with 18.6% efficiency via a through‐bond mechanism, resulting in the isomerization of the latter group to the quadricyclane group.

Benzophenone-Initiated Photoisomerization of the Norbornadiene Group in a Benzophenone−Steroid−Norbornadiene System via Long-Distance Intramolecular Triplet Energy Transfer

Bichromophoric compound 3β-((2-(methoxycarbonyl)bicyclo[2.2.1]hepta-2,5-diene-3-yl)carboxy)androst-5-en-17β-yl benzophenone-4-carboxylate (NBD-S-BP) was synthesized, and its photochemistry was examined. The phosphorescence of the benzophenone (BP, donor) chromophore is efficiently quenched by the remote norbornadiene (NBD, acceptor) group. Time-resolved spectroscopic measurements indicate that the lifetime of the triplet state of the BP chromophore is shortened by the NBD group. Selective excitation of the BP chromophore results in isomerization of the NBD group to quadricyclane. All these observations suggest that a long-distance intramolecular triplet energy transfer occurs in the NBD-S-BP molecule. The efficiency and rate constant for this triplet energy transfer were determined to be ca. 22% and 1.5 × 105 s-1, respectively, by steady-state photolysis and laser flash photolysis. This long-distance intramolecular triplet energy transfer is proposed to proceed via a through-bond exchange mechanism.

Dipole moment change of NBD group upon excitation studied using solvatochromic and quantum chemical approaches: Implications in membrane research

Lipids that are covalently labeled with the 7-nitrobenz-2-oxa- 1,3-diazol-4-y1 (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. We have recently shown that one such NBD-labeled lipid, NBD-PE, in which the NBD label is covalently attached to the headgroup of a phosphatidylethanolamine molecule, exhibits the red edge excitation shift (REES) effect when incorporated into vesicles of dioleoyl-sn-glycero-3-phosphocholine(D OPC) [Chattopadhyay, A,; Mukherjee, S. Biochemistry 1993,32,3804]. One of the necessary conditions for a fluorophore to be able to exhibit REES is that the fluorophore must be polar and, more importantly, there should be a change in its dipole moment upon excitation. In this paper, we have determined the actual change in dipole moment of the NBD group upon excitation using the solvatochromic shift approach. Our results show that the dipole moment of the NBD group changes by 3.9 D upon excitation. We have complemented this experimental observation by semiempirical quantum chemical calculations of dipole moment changes of various NBD derivatives. These calculated dipole moment changes (3.5-3.6 D) agree very well with our experimental value. These calculations also point out that the process of charge separation is mainly limited to the NBD ring system and is independent of the length of the alkyl chain. These results are relevant to ongoing and future studies that utilize photophysical properties of the NBD group, especially in microheterogeneous media such as membranes and micelles.

Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K +-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 × 10 5 g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K +-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 m KCl (6 S elongated state) when compared to that at 0.15 m KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.

Modification of the (Ca2 + Mg2)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.

Transfer of phosphatidic acid from liposomes to cells is collision dependent.

The kinetics of lipid transfer from unilamellar liposomes to cells in monolayer culture were determined for a fluorescent phosphatidic acid, 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] -sn-glycerol 3-phosphate (C6-NBD-PA), and for the analogous phosphatidic acid without the fluorescent NBD group, 1-palmitoyl-2-caproyl-sn-[U-14C] glycerol 3-phosphate (C6-[14C]PA). Initial rates of liposome-to-cell transfer were measured at 2 degrees C under conditions in which the concentration of diffusible monomer in the aqueous medium was constant during the course of an experiment and was independent of total liposome concentration. Rates were similar for C6-NBD-PA and C6-[14C]PA, indicating that the NBD group does not significantly alter the transfer kinetics. It was found that liposome-to-cell transfer was dependent on 1) the mole fraction of diffusible lipid in the liposomes, 2) the liposome concentration, and 3) the cell density. The dependence of rate on the liposome concentration (observed under conditions in which aqueous monomer concentration remained constant) cannot be explained by a liposome-to-cell transfer mechanism involving the free diffusion of monomers through the aqueous medium. Instead, the data are consistent with a collision-dependent mechanism of monomer transfer that occurs when liposome and cell membranes come into contact but do not fuse.

Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- labeledlipidsinmodelmembranes

The spectroscopic and ionization properties of various lipids labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group have been studied in model membranes using fluorescence, absorbance and electrophoretic mobility measurements. Electrophoretic measurements show that the NBD group is uncharged at neutral pH. However, at high pH, hydroxyl addition or deprotonation occurs with a p K a , depending upon conditions, of 11.5–11.8 for the NBD group of headgroup-labeled phosphatidylethanolamine (NBD-PE) and 11.1–11.5 for NBD labels placed at the end of one fatty acyl chain of a phosphatidylcholine (6-NBD-PC and 12-NBD-PC). This type of behavior is not observed in the case of a methylated NBD label placed in the flexible ‘tail’ of cholesterol (NBD-cholesterol). The similarity in p K a for NBD-PE and NBD-PCs suggests that in these cases the NBD group is at a similar depth in the membrane. This was examined further by comparison of the fluorescence emission maximum of the NBD group in model membranes with that in solvents of varying polarity. The apparent polarity experienced by NBD groups in model membranes indicates that for NBD-PE and 12-NBD-PC they are located at the polar region whereas the NBD group of NBD-cholesterol is deeply buried in a nonpolar region of the membrane. This conclusion is supported further by fluorescence quenching experiments measuring NBD exposure to the aqueous quencher Co 2+. The results of this study confirm the tentative conclusions of our previous fluorescence quenching studies on the location of NBD groups in model membranes.

Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids.

This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of depth in angstroms. Furthermore, the analysis can be extended to quenching by energy-transfer acceptors or brominated probes under appropriate conditions. Application of the method to quenching of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipids by spin-labeled lipids located at three different depths is demonstrated in model membranes. It is shown that the calculated depths of the NBD groups are self-consistent to the extent that they are the same no matter which two spin-labels have been used in a particular experiment. In addition, the calculated depth is independent of spin-label concentration in the membrane within +/- 1 A, ruling out major effects due to spin-label perturbation. The quenching experiments show that the location of the NBD group in head-group-labeled phosphatidylethanolamine is at the polar/hydrocarbon interface and that of an NBD label on the "tail" of cholesterol is deeply buried, as expected. Unexpectedly, NBD labels placed at the end of fatty acyl chains of phosphatidylcholines are also near the polar/hydrocarbon interface. Presumably, the polarity of the NBD group results in "looping" back to the surface of the NBD groups attached to flexible acyl chains.