Uptake of N-(9-Anthroyloxy) Fatty Acid Fluorescent Probes Into Lipid Bilayers

Haigh, Elizabeth A; Thulborn, Keith R.; Nichol, L.W.; Sawyer, William H.

doi:10.1071/bi9780447

Cited by 31 publications

(10 citation statements)

References 12 publications

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“…These were, expressed as percentages of the 2AF value, 100% (2AF), 96% (3AF), 7 8 8 (6AF), 70% (7AF), 61% (9AF), 49% (12AF), and 47% (16AF). This curious correlation of decreasing amount of uptake with increasing n-number has been noted previously (7)(8)(9)14). Figure 1B shows the same data expressed as percentage of quenching and indicates that the order of quenching is, with respect to n, 2 2 3 > 6 2 7 > 9 > 12 > 16.…”

Section: Resultssupporting

confidence: 79%

“…The decreasing amount of uptake with increasing n-number ( Fig. IA) has been noted previously and suggested to be due to the number of cellular receptors (14) or to the size of the probe aggregates in the dispersals added to the cells (7,8). We discount the possibility that it is solely a consequence of probe localization into the membrane (i.e., the hy-drocarbon chain "below" the anthroyloxy group would penetrate with relative ease, whereas it would take longer for penetration of the bulky anthracene ring), as a t equilibrium, the amounts of uptake would be similar, and they are not (see ordinate values a t zero abscissa, Fig.…”

Section: Discussionsupporting

confidence: 69%

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Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Collins

Grogan

1991

Cytometry

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The transverse location normal to the bilayer surface of a series of n-(g-anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cuc2, was 2 2 3 > 6 2 7 > 9 > 12 > 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells. Key terms: Anthroyloxy fatty acids, membrane fluidity, HeLaThe introduction by Waggoner and Stryer (22) of a fluorescent fatty acid, 12-(9-anthroyloxy) stearate, as a membrane probe able to locate a t a defined depth, offered a potentially useful approach to understanding the dynamics of the outer hemi-leaflet of the membrane bilayer; other members of the n-AF, or n-(9-anthroyloxy) fatty acid, series quickly followed. At the outset these probes were expected to tether their negatively charged carboxyl groups a t the surface in contact with the aqueous phase, with the anthroyloxy moieties thus locating a t different depths. There is presently little doubt that the n-AF probes indeed locate at different depths in model lipid bilayers. In dipalmitoylphosphatidyl lecithin vesicles, NMR spectra detected a chemical shift near the first and second methylene groups of the lipid acyl chains with 2-(9-anthroyloxy) palmitate, thus placing the absolute position of this anthroyloxy group level with the acyl chain's ester linkage, near the surface (19). With 12-(9-anthroyloxy) stearate, the shift occurred for the terminal methylene groups of the acyl chains, thus placing that, anthroyloxy group nearer the bilayer center (19). In dipalmitoylphosphatidyl choline bilayers, quenching agents of fluorescence, which locate at the surface or a t the bilayer center, revealed that 2-AF had portions of the anthroyloxy moiety in both the aqueous and the lipid phases, whereas 6-AF was entirely in the lipid phase (21). In model lipid bilayers, with spin-labeled fatty acids as paramagnetic quenchers, the order of quenching with respect to alkyl chain position, n, was always 2 > 6 > 9 > 12 > 16 (1). The aqueous quenching of anthroyloxy fluorescence in isolated beef mitochondria by C u f " was in the order, 2 > 6 > 9 > 12 > 16, whereas with dimethylaniline, a quencher that partitions into the lipid phase near the bilayer center, the order was reversed (5).Therefore, the most reasonable interpretation of these studies is that the n-AF probes appear to locate in a graded series of depths from the surface to the center of the lipid bilayer, at least in model systems. However, this has never been demonstrated with intact, living cells. The recent adaptation of commercial flow cytometers for pol...

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Section: Resultssupporting

confidence: 79%

Section: Discussionsupporting

confidence: 69%

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Collins

Grogan

1991

Cytometry

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“…This be- haviour differs from the rectangular hyperbola observed with other proteins [21,27,281. A linear increase in fluorescence with increasing fluorophore is indicative of partitioning [29]. The data shown in Fig.…”

Section: Resultsmentioning

confidence: 92%

Binding of 1‐Anilinonaphthalene‐8‐Sulfonic Acid to α‐crystallin

Stevens¹,

Augusteyn²

1997

European Journal of Biochemistry

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a-Crystallin was found to exhibit a time-dependent uptake of the hydrophobic probe, l-anilinonaphthalene-8-sulfonic acid (ANS), similar to that typically observed with lipid membranes. Analysis of the interaction of ANS with a-crystallin revealed two types of interactive processes, partitioning and binding. The predominant process involved partitioning, with a coefficient of 300 M-'. The binding component had the following characteristics: 1 binding site124 subunits and a Kd of about 9 pM. The binding was unaffected by the number of subunits used in the assembly of the a-aggregate, since both the an,-and a,-forms had similar binding characteristics. No discernible differences were observed in the binding of ANS to homopolymers of aA and aB subunits, suggesting that the hydrophobic sites to which ANS bound were similar in both the A and B subunits. The majority of the fluorescence was lost when the protein was incubated in 3 M urea, a concentration of denaturant where the protein is still intact, suggesting that the ANS binding sites are located near the surface of the protein. The decrease was attributed to a decrease in the quantum yield of the bound dye.Keywords: a-crystallin; I-anilinonaphthalene-8-sulfonic acid; quaternary structure; hydrophobic interaction.a-Crystallin constitutes up to 50% of the total proteins which comprise the bulk of the mammalian eye lens. Its relatively constant proportion in most species suggests that it has an important role in the lens. The absence of any tissue turnover necessitates that it remains functional for the lifetime of the organism, in the case of humans in excess of 70 years. In cases where the protein is modified or structurally disrupted impairment of vision results, usually in the form of a cataract. Recently, it has been shown that a-crystallin is a member of the small heat-shockprotein family and may function as a chaperone [I]. It has been suggested that one of its major functions is to prevent age-related aggregation of lens proteins, thereby maintaining lens transparency [2]. The mechanisms for this function have not been delineated but they probably involve the surface of a-crystallin which has been shown to be very hydrophobic [3, 41. Knowledge of the location of these hydrophobic sites would be of enormous benefit in determining how a-crystallin functions as a molecular chaperone, as well as providing insight into its structure.a-Crystallin is a polymeric protein which consists of two types of subunits, A and B, both of which have a mass around 20 kDa [5]. Their amino acid sequences are closely related and have changed very slowly during the course of evolution, reflecting a structural constraint on the protein. This also suggests that it has an important role in maintaining interactions necessary for the formation of a transparent lens. Both polypeptides are subject to various chemical and enzymatic modifications such as oxidation, phosphorylation, deamidation, glycation, and C-terminal degradation 161. These modifications are thought to

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“…The orientation of the fluorescent probes in membranes has been best studied by following the depth-dependent quenching of their fluorescence in membranes (Blatt & Sawyer, 1985). The orientation of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12) in artificial membranes has been studied by using this technique (Thulborn, 1981;Haigh et al, 1979). To get an insight into the orientation of fluorenyl fatty acids in PC vesicles, we have studied their fluorescence quenching in vesicles using extrinsic water-soluble quenchers like iodide and Cu(I1) and intrinsic lipid-soluble quenchers like 12-bromostearic acid and 9,lOdibromostearic acid.…”

Section: Resultsmentioning

confidence: 99%

Design, synthesis, and fluorescence studies of fluorenyl fatty acids as new depth-dependent fluorescent probes for membranes: getting over the looping-back problem

Lala

Dixit²,

Koppaka³

et al. 1988

Biochemistry

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Fluorescent fatty acids have proved very useful in studying the membrane hydrophobic core. They readily partition into membranes or can be converted to phospholipids, which form integral components of membranes. By attaching the fluorescent chromophore to different positions along the alkyl chain of fatty acids, e.g., an anthroyloxy group attached via an ester linkage to n-hydroxystearic acid, membranes have been probed at different depths. While this is an interesting approach and has been extensively used, relatively little attention has been paid to the molecular design of these probes in order to have minimal membrane perturbation. In the present study we have looked into the general problem of design of such depth-dependent membrane probes. We report here a series of fluorenyl fatty acids with varying fatty acid chain lengths, Le., (2-fluoreny1)acetic acid, -butyric acid, -hexanoic acid, and -octanoic acid, in order to obtain information at different depths in the membrane hydrophobic core. To see the effect of attachment of a hydrophobic tail on the orientation of such fatty acids in membranes, an n-butyl group was linked to the C-7 position of fluorene in (2-fluoreny1)butyric acid to get 4-(7-n-butylfluoren-2-yl)butyric acid. Further, to assess their ability to act as depth-dependent fluorescent probes, these fatty acids were incorporated in vesicles prepared from egg phosphatidylcholine, and their fluorescence quenching was studied with potassium iodide, Cu(II), 9,lO-dibromostearic acid, and 12-bromostearic acid. The Stern-Volmer plots so obtained clearly indicated the following depth order: 8-(2-fluorenyl)octanoic acid > 6-(2-fluorenyl)hexanoic acid > 4-(7-n-butylfluoren-2-yl)butyric acid > 4-(2-fluorenyl)butyric acid > (2-fluoreny1)acetic acid. These results indicate that the fluorenyl fatty acids reported here do probe the membrane at different depths, depending on their transverse location. In addition, the attachment of a hydrophobic tail helps in better aligning the probe in membranes and could thus overcome the problems associated with looping back of such fatty acids to the membrane-water interface. These results were also supported by fluorescence polarization data. The general design strategy utilized here can in principle be extended to other membrane probes like photoactivatable fatty acids.

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Uptake of N-(9-Anthroyloxy) Fatty Acid Fluorescent Probes Into Lipid Bilayers

Cited by 31 publications

References 12 publications

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Binding of 1‐Anilinonaphthalene‐8‐Sulfonic Acid to α‐crystallin

Design, synthesis, and fluorescence studies of fluorenyl fatty acids as new depth-dependent fluorescent probes for membranes: getting over the looping-back problem

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