Shearwater Foraging in the Southern Ocean: The Roles of Prey Availability and Winds

Background

Sooty (Puffinus griseus) and short-tailed (P. tenuirostris) shearwaters are abundant seabirds that range widely across global oceans. Understanding the foraging ecology of these species in the Southern Ocean is important for monitoring and ecosystem conservation and management.

Methodology/Principal Findings

Tracking data from sooty and short-tailed shearwaters from three regions of New Zealand and Australia were combined with at-sea observations of shearwaters in the Southern Ocean, physical oceanography, near-surface copepod distributions, pelagic trawl data, and synoptic near-surface winds. Shearwaters from all three regions foraged in the Polar Front zone, and showed particular overlap in the region around 140°E. Short-tailed shearwaters from South Australia also foraged in Antarctic waters south of the Polar Front. The spatial distribution of shearwater foraging effort in the Polar Front zone was matched by patterns in large-scale upwelling, primary production, and abundances of copepods and myctophid fish. Oceanic winds were found to be broad determinants of foraging distribution, and of the flight paths taken by the birds on long foraging trips to Antarctic waters.

Conclusions/Significance

The shearwaters displayed foraging site fidelity and overlap of foraging habitat between species and populations that may enhance their utility as indicators of Southern Ocean ecosystems. The results highlight the importance of upwellings due to interactions of the Antarctic Circumpolar Current with large-scale bottom topography, and the corresponding localised increases in the productivity of the Polar Front ecosystem.     

Differences in cell viabilities of phytoplankton between spring and late summer in the northwest Pacific Ocean

Cell viabilities of phytoplankton in the Oyashio and Kuroshio-Oyashio transition regions of the northwest Pacific Ocean were examined in September 2003 (late summer) and May 2005 (spring) using a membrane permeability test. Specific lysis rates of the phytoplankton during late summer were also assessed by an esterase activity assay. In late summer, cyanobacteria Synechococcus spp. were > 2 × 10⁴ cells ml^− 1 and numerically dominated the phytoplankton communities. The cell viabilities of Synechococcus spp. and eukaryotic ultraphytoplankton (< 10 μm in size) were 60-79% and 26-41% in surface waters, respectively. The specific lysis rates of the phytoplankton were 0.12-0.67 d^− 1 in late summer. By contrast, in spring, eukaryotic cells were predominant in the phytoplankton communities. The cell viabilities of surface eukaryotic ultraphytoplankton in spring were > 70% and significantly higher than those in late summer. During spring, Synechococcus spp. only occurred with < 1 × 10⁴ cells ml^− 1 in the Kuroshio-Oyashio transition region, and their viabilities were 80%. In the Oyashio region where a spring diatom bloom developed, the viability of fucoxanthin-containing algae (mainly diatoms and prymnesiophytes) was ca. 90%. These results suggested that the cell viability of phytoplankton could vary seasonally with their community structure in the study area. The phytoplankton cell death in late summer was particularly significant for their loss process and could support the microbial food webs by supplying dissolved organic carbon (DOC) derived from the dead cells.     

Distribution of living coccolithophores in the California Current system,southern California borderland

The distribution of living coccolithophores in the California Current system of southern California at 10 m water depth was investigated on two dates in March and June, 1982. Six closely spaced stations were sampled in March, of which three were resampled in June. Thirty-six euphotic species were identified of which four,Emiliania huxleyi, Umbilicosphaera sibogae, Gephyrocapsa oceanica, andRhabdosphaera longistylis, respectively, were the most abundant. Both the “cold” and “warm” morphotypes ofE. huxleyi were present, in varying proportions. Large ranges in community structure, diversity (0.35–2.64 natural bels), and standing crop (1.0 × 10⁴–6.2 × 10⁵cells/l) were recorded. This range of end-member values is approximately that found in the open ocean from 0° to about 65° latitude.The distributions of four coccolithophore assemblages recognized in March samples from the Borderland area appear to reflect the following distinct water masses: (1) California Current; (2) Southern California Counter Current; (3) Transitional Zone; (4) Near Shore. The coccolithophore assemblages from the June stations were more uniform, indicating that the Borderland was experiencing more stable conditions than in March.     

Pigment distribution in the Pacific region of the Southern Ocean (autumn 1995)

Phytoplankton distribution patterns are still largely unknown for the Pacific region of the Southern Ocean. Pigment distributions were determined by HPLC on 40-m samples collected from the mixed layer during the ANTXII/4 cruise in March–May 1995 aboard RV “Polarstern”. A transect was covered (90°W, from 51°S to 70°S), crossing the Subantarctic Front in the north, the Polar Front, and the Southern Polar Front in the south. Coinciding with high concentrations of silicate, diatoms dominated in the Antarctic waters south of the Polar Front. North of the Polar Front, silicate concentrations dropped to values less than 10 μM. In this area flagellates (Prymnesiophyceae and green algae) were the dominant phytoplankton group. Nutrient depletion of the surface waters near the Southern Polar Front indicated formerly enhanced productivity. These findings confirmed previous observations by the British Sterna expedition, which described locally elevated chlorophyll a biomass near the southern boundary of the Southern Polar Front. We propose a role for supply of bioavailable iron via the front, and emphasise the importance of frontal systems for phytoplankton productivity in the Southern Ocean. Received: 11 June 1997 / Accepted: 16 November 1997     

Quantitative analysis of the bacteriophage Qβ infection cycle

In this study, the infection cycle of bacteriophage Qβ was investigated. Adsorption of bacteriophage Qβ to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4 × 10^− 10 ml/cells/min. In infected cells, approximately 130 molecules of β-subunit and 2 × 10⁵ molecules of coat protein were translated in 15 min. Replication of Qβ RNA proceeded in 2 steps—an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5 × 10⁵ molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.     

Effects of temperature and salinity on the standard metabolic rate (SMR) of the caridean shrimp Palaemon peringueyi

The standard metabolic rate (SMR) of the caridean shrimp Palaemon peringueyi to changes in temperature (15-30 °C), salinity (0-45‰) and a combination thereof was investigated. The rate of oxygen consumption of the shrimp was determined using a YSI oxygen meter. At a constant salinity of 35‰ the respiration rate of P. peringueyi increased with an increase in temperature and ranged between 0.260 and 0.982 μl O₂ mg wwt^− 1 h^− 1. The Q₁₀ value over the temperature range 15-25 °C was estimated at 3.13. At a constant temperature of 15 °C the respiration rate of P. peringueyi also increased with an increase in salinity and ranged between 0.231 and 0.860 μl O₂ mg wwt^− 1 h^− 1. For combination experiments the absence of any significant difference in the respiration rate of P. peringueyi at the four temperatures over the salinity range 15-35‰ suggests that the shrimp is well adapted to inhabiting environments characterised by variations in salinity and temperature such as those encountered within the middle and lower reaches of permanently open estuaries with substantial freshwater inflow. On the other hand, the total mortality of the shrimp recorded at salinities < 5‰ at all four temperatures suggests that the upper distribution of the shrimp may reflect physiological constraints. Similarly, the increase in the respiration rate of the shrimp at the four temperatures at salinities > 35‰ suggests that the shrimp may experience osmotic stress in freshwater deprived permanently open and intermittently open estuaries where hypersaline conditions may develop.     

Oligomeric-State-Dependent Conformational Change of the BLUF Protein TePixD (Tll0078)

The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 μs, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 × 10^− 11 to 4.4 × 10^− 11 m² s^− 1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10^− 11 m² s^− 1 upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.     

Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K₁ = 21 ± 6 × 10³ M^− 1) than the second (K₂ = 4 ± 2 × 10³ M^− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K₁ = 20 ± 10 × 10³ M^− 1 and K₂ = 1.67 ± 0.07 × 10³ M^− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.     

Influence of cell equivalent spherical diameter on the growth rate and cell density of marine phytoplankton

The maximal growth rates (μ_max) of 8 species of marine phytoplankton were studied in detail. A Logistic growth model was used to describe the growth process of phytoplankton and the averaged plotting correlation coefficient was 1.00 ± 0.01 (mean ± standard deviation). The size distribution of phytoplankton could be well represented by single or combined Gaussian distribution functions. The size distribution of phytoplankton was investigated by daily analysis, and the variation of the median equivalent spherical diameter (MESD) was recorded. The size of algal cells varied throughout the process of population growth, and the size distribution characteristic of the two species of pyrrophytes investigated also changed during the growth process. The relation between maximum growth rate and MESD could be expressed by the equation μ_max = a * MESD^b (where μ_max is the maximum specific growth rate, MESD is the median equivalent spherical diameter, and a and b are constants equal to 2.10 × 10⁵ and − 1.15, respectively), estimated by nonlinear regression analysis with the allometric function. The dependence of maximum cell density on algal MESD was also investigated and the relationship B_f = 1.56 × 10⁷ MESD^− 1.20 was obtained (where B_f is the maximum cell density).     

Light-induced conformational change and transient dissociation reaction of the BLUF photoreceptor Synechocystis PixD (Slr1694)

The light-induced reaction of the BLUF (blue light photoreceptor using flavin adenine dinucleotide) photoreceptor PixD from Synechocystis sp. PCC6803 (Slr1694) was investigated using the time-resolved transient grating method. A conformational change coupled with a volume contraction of 13 mL mol^− 1 was observed with a time constant of 45 ms following photoexcitation. At a weak excitation light intensity, there were no further changes in volume and diffusion coefficient (D). The determined D-value (3.7 × 10^− 11 m² s^− 1) suggests that PixD exists as a decamer in solution, and this oligomeric state was confirmed by size-exclusion chromatography and blue native polyacrylamide gel electrophoresis. Surprisingly, by increasing the excitation laser power, we observed a large increase in D with a time constant of 350 ms following the volume contraction reaction. The D-value of this photoproduct species (7.5 × 10^− 11 m² s^− 1) is close to that of the PixD dimer. Combined with transient grating and size-exclusion chromatography measurements under light-illuminated conditions, the light-induced increase in D was attributed to a transient dissociation reaction of the PixD decamer to a dimer. For the M93A-mutated PixD, no volume or D-change was observed. Furthermore, we showed that the M93A mutant did not form the decamer but only the dimer in the dark state. These results indicate that the formation of the decamer and the conformational change around the Met residue are important factors that control the regulation of the downstream signal transduction by the PixD photoreceptor.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Mycosporine-like amino acids in azooxanthellate and zooxanthellate early developmental stages of the soft coral Heteroxenia fuscescens

The ontogenetic changes of MAAs in the soft coral Heteroxenia fuscescens was studied in relation to their symbiotic state (azooxanthellate vs. zooxanthellate) under different temperature conditions in the Gulf of Eilat, northern Red Sea. The HPLC chromatograms for extracts of the planulae, azoo- and zooxanthellate primary polyps of H. fuscescens from all dates of collection yielded a single peak at 320 nm that has been identified as the compound palythine. Concentration of palythine in planulae at 23 °C was 7.57 ± 1 nmol mg^− 1 protein and at 28 °C reached 17.29 ± 1 nmol × mg^− 1 protein. Concentration of palythine in azooxanthellate primary polyps was 16.4 ± 3 nmol × mg^− 1 protein and 28.37 ± 2.8 nmol × mg^− 1 protein at 23 °C and 28 °C respectively. The palythine concentration for zooxanthellate primary polyps at 23 °C was 13 ± 3 nmol × mg^− 1 protein and at 28 °C 32.7 ± 2 nmol mg^− 1 protein. Palythine concentrations were significantly higher at 28 °C in the different animal groups and correlated linearly with the ambient collection temperature. This study shows for the first time that UVR and temperature act synergistically and affect the MAA levels of early life-history stages of soft corals.     

Near anoxia and sulfide as possible factors influencing the spatial distribution of Acartia tonsa and Acartia clausi: Comparative evaluation of egg tolerance

In many coastal and estuarine areas the planktonic copepods Acartia tonsa and Acartia clausi show a spatial separation with A. tonsa restricted to brackish waters and confined environments and A. clausi inhabiting areas more influenced by sea water. The hatching and viability of A. tonsa and A. clausi eggs exposed to anoxia and anoxia/sulfide (conditions that are frequent in bottom waters of the most confined areas) was evaluated to determine if these stress factors play a role in the distribution of these species. Subitaneous eggs, spawned by laboratory reared organisms, were incubated in near anoxia (< 7.59 × 10^− 3 mmol O₂ L^− 1) or anoxia/sulfide (∼ 1 mmol L^− 1) for different periods (1, 4, 8 and 15 days), then transferred to normoxic conditions. The exposure of the eggs to near anoxia and sulfide appears to induce the same response (quiescence) in both species. Exposure times ≤ 8 days to near anoxia or anoxia/sulfide did not affect egg viability, while 15 day exposure caused significant declines in hatching success of both species. A significant difference between the effects of near anoxia and anoxia/sulfide was observed when incubation lasted 15 days; hatching of eggs exposed to sulfide being higher than that of eggs exposed to near anoxia for both species. No significant differences were observed between the two species in hatching success of eggs exposed to both near anoxia and anoxia/sulfide (with the exception of eggs incubated in near anoxia for 4 days). The results indicate that the impact of anoxia and sulfide on the eggs of the two Acartia species cannot be a factor explaining the spatial distribution in coastal and brackish environments of these copepods.Feeding experiments on A. clausi were also performed. Suitability of different algal species to rear this copepod was evaluated and the results were compared with data previously obtained for A. tonsa. Differences in feeding needs between A. clausi and A. tonsa are discussed.     

Characterisation of the electron transfer and complex formation between Flavodoxin from D. vulgaris and the haem domain of Cytochrome P450 BM3 from B. megaterium

Investigation of the complex formation and electron transfer kinetics between P450 BMP and flavodoxin was carried out following the suggested involvement of flavodoxin in modulating the electron transfer to BMP in artificial redox chains bound to an electrode surface. While electron transfer measurements show the formation of a tightly bound complex, the NMR data indicate the formation of shortly lived complexes. The measured k_obs ranged from 24.2 s^− 1 to 44.1 s^− 1 with k_on ranging from 0.07 × 10⁶ to 1.1 × 10⁶ s^− 1M^− 1 and K_d ranging from 300 μM to 24 μM in buffers of different ionic strength. This apparent contradiction is due to the existence of two events in the complex formation prior to electron transfer. A stable complex is initially formed. Within such tightly bound complex, flavodoxin rocks rapidly between different positions. The rocking of the bound flavodoxin between several different orientations gives rise to the transient complexes in fast exchange as observed in the NMR experiments. Docking simulations with two different approaches support the theory that there is no highly specific orientation in the complex, but instead one side of the flavodoxin binds the P450 with high overall affinity but with a number of different orientations. The level of functionality of each orientation is dependent on the distance between cofactors, which can vary between 8 and 25 Å, with some of the transient complexes showing distances compatible with the measured electron transfer rate constants.     

Assimilation and oxygen uptake associated with two different feeding habits of Munida gregaria (=M. subrugosa) (Crustacea, Decapoda)

Munida gregaria is the most abundant galatheid crab species in the Beagle Channel (55 °S 68 °W) off Tierra del Fuego, Argentina. This species has two different and simultaneous feeding habits: predator and deposit feeder. The objectives of this work were to determine whether either of the two different feeding habits imply differences in assimilation and oxygen consumption. Subsamples of 40 and 20 crabs of each sex were randomly selected, transported to the laboratory, and used in assimilation and oxygen consumption experiments, respectively. Animals were placed in individual chambers at 8 ± 1 °C. The assimilation experiment was conducted using four types of diet: isopods (CRU), algae (ALG), pulverized fish food (PFF), and particulate organic matter (POM). The crabs' routine metabolism and postprandial oxygen consumption - or specific dynamic action SDA - were measured using Clark-type polarographic electrodes. Two kinds of food were offered: CRU and PFF to simulate both feeding habits. Assimilation in M. gregaria for both feeding habits presented values > 90% for dry mass and energetic content. Consumption rate varied between 15 and 50 mg_ingested g_DM^− 1, and was significantly higher for CRU and PFF diets. Females showed significantly higher consumption rates than males for all diets. On average, the routine metabolism of M. gregaria was 15 ± 5 μlO₂ h^− 1 g^− 1. Deposit feeding may confer energetic advantages to female M. gregaria, especially because this diet has a low energetic cost of assimilation. Deposits of high-energy content showed the highest consumption rates among four the experimental diets. In terms of mass and energy, the deposit diet also showed the highest assimilation efficiency. The postprandial oxygen consumption was lower in females and in the deposit feeding experiment. We suggest that females may prefer the deposit feeding habit to maximize their ingested energy, allowing them to devote more energy to reproduction. This is attained by a high consumption rate and by minimizing the energy used in assimilation.     

The impact of ice melting on bacterioplankton in the Arctic Ocean

Global warming and the associated ice melt are leading to an increase in the organic carbon in the Arctic Ocean. We evaluated the effects of ice melt on bacterioplankton at 21 stations in the Greenland Sea and Arctic Ocean in the summer of 2007, when a historical minimum of Arctic ice coverage was measured. Polar Surface Waters, which have a low temperature and low salinity and originate mainly from melted ice, contained a very low abundance of bacteria (7.01 × 10⁵ ± 2.20 × 10⁵ cells ml⁻¹); however, these bacteria had high specific bacterial production (2.40 ± 1.61 fmol C bac⁻¹ d⁻¹) compared to those in Atlantic Waters. Specifically, bacterioplankton in Polar Surface Waters showed a preference for utilizing carbohydrates and had significantly higher specific activities of the glycosidases assayed, i.e. β-glucosidase, xylosidase, arabinosidase and cellobiosidase. Furthermore, bacterioplankton in Polar Sea Waters showed preferential growth on some of the carbohydrates in the Biolog Ecoplate, such as d-cellobiose and N-acetyl-d-glucosamine. Our results suggest that climate change and the associated melting of Arctic ice might induce changes in bacterioplankton functional diversity by enhancing the turnover of carbohydrates. Since organic aggregates are largely composed of polysaccharides, higher solubilization of aggregates might modify the carbon cycle, weaken the biological pump and have biogeochemical and ecological implications for the future Arctic Ocean.     

Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (K_a) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average K_a values of 1.3 (±0.2) × 10⁵ and 3.5 (±3.0) × 10² M⁻¹ for acetohexamide and values of 8.7 (±0.6) × 10⁴ and 8.1 (±1.7) × 10³ M⁻¹ for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with K_a values of 1.3 (±0.1) × 10⁵ and 4.3 (±0.3) × 10⁴ M⁻¹, respectively, at 37 °C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with K_a values of 5.5 (±0.2) × 10⁴ and 5.3 (±0.2) × 10⁴ M⁻¹, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug–protein interactions.     

Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs

It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC (k_dis = 0.115 s^− 1; t_1/2 = 6.03 s); Bodipy FL-PC (k_dis = 5.2 × 10^− 4; t_1/2 = 22.2 min); Bodipy 530-PC (k_dis = 1.52 × 10^− 5; t_1/2 = 12.6 h); and Bodipy 581-PC (k_dis = 5.9 × 10^− 6; t_1/2 = 32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.