Interplay of Secondary Structure and Charge on the Diffusion of a Polypeptide through Negatively Charged Aqueous Pores (original) (raw)
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International journal of …, 2002
The purpose of the present study was to investigate the effect of secondary structure of three model polypeptides on their apparent permeability (P app ) across a synthetic, microporous membrane. Poly-L-lysine (PL), poly-L-glutamate (PGlu), and poly-L-lysine-L-phenylalanine (1:1) (PLP) were selected because a solution environment in which their predominant secondary structure is random coil (RC), a-helix, and b-sheet, respectively, is easily achieved. The conformation of each polypeptide was verified by circular dichroism (CD). Diffusion studies were conducted under sink conditions at 25°C across a microporous polyester membrane using a donor concentration of 0.02 mM for each model polypeptide. NMR was utilized to obtain a second estimation of the diffusion coefficient for each polypeptide. The equivalent hydrodynamic radii (R e ) of the three model polypeptides were calculated using the values of the diffusion coefficient obtained by both NMR and the classic in vitro diffusion studies. The viscosity of each polypeptide solution was also determined to investigate the effect of viscosity on the aqueous diffusion coefficient. Statistical analysis demonstrated a significant (P B0.05) difference in both P app and the aqueous diffusion coefficient (D aq ), as well as the calculated R e values, between all three model polypeptides and there was no significant (P \ 0.05) difference in the viscosity of the polypeptide solutions. Values of D aq and R e calculated from the diffusion studies were in relatively close agreement to those obtained using NMR. The logarithm of P app was highly correlated (r= −0.961) with the values of R e calculated from NMR (R e (NMR) ) rather than the mw of the polypeptides (r= 0.681). Values of the Perrin or shape factor which deviate substantially from unity are suggestive of a non-spherical or ellipsoid shape and were 1.22 90.20, 1.55 9 0.11, and 2.38 9 0.20 for PGlu, PL, and PLP, respectively. In conclusion, the observed difference in the membrane transport/diffusion of the three model polypeptides is suggested to be due to the shape associated with the secondary structure of each macromolecule, rather than the polypeptide's mw or the viscosity of the dilute polypeptide solution.
Molecular mobility and transport in polymer membranes and polyelectrolyte multilayers
Magnetic Resonance Imaging, 2007
Polyelectrolyte multilayers prepared by the layer-by-layer technique provide an efficient way to generate planar structures of tailored surface charge and hydrophobicity, which are used as membranes for pervaporation. The use of polyelectrolyte multilayers to form the membrane permits tailoring the surface charge of the membrane and, thus, selectivity; at the same time, it reduces fouling of the membrane by adsorption of organic matter. Pulsed field gradient (PFG) nuclear magnetic resonance has been used to investigate the diffusion of probe molecules into polymer systems. Evaluation of the apparent diffusion coefficient in porous poly(amide) results in a pore size of 4 Am, as found in electron micrographs. For the pore size obtained for polyelectrolyte multilayers, no equivalent pores could be found in microscopy. Propagators for the diffusion of propanol and propanol-water mixture into multilayers reveal that there might be selective interaction of probe molecules with the polyelectrolyte system. D
Journal of Pharmaceutical Sciences, 1997
We applied the principles of molecular-size-restricted diffusion within a negative electrostatic field of force to follow the changes in the aqueous pore radius of tight junctions (TJs) induced by perturbants and the accompanying influence on the permeation of neutral (urea and mannitol), cationic (methylamine and atenolol), and anionic (formate and lactate) compounds that vary in size. The perturbants included palmitoyl-DL-carnitine (PC), which opens TJs by an unknown Ca ++-independent mechanism, and ethyleneglycol-bis-(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), a Ca ++ chelator. Mass transfer resistances of the collagen−coated filter support and the aqueous boundary layers were factored out to yield paracellular permeability coefficients (P P). As viewed from the P P values of urea and mannitol, EGTA exhibited insignificant effects on pore size at low concentrations compared with control, and then caused a dramatic opening of the TJs over a narrow concentration range (1.35−1.4 mM). The P P values for urea and mannitol remained constant at >1.4 mM EGTA. However, PC produced dose−dependent responses from 0 to 0.15 mM that plateaued at >0.15 mM. In general, cations permeated the cellular TJs faster and anions slower than their neutral images. The effects of changes in pore size (4.6 to 14.6 Å in effective radius) on the ability of these solutes to permeate the TJs were analyzed by the Renkin molecular sieving function. These studies established an experimental, theoretical, and quantitative template to assess perturbants of the TJ and define the limits, short of detrimental effects, at which the TJs may be sufficiently perturbed for maximal enhancement of permeation of solutes varying in size and charge. Materials and Methods MaterialssMannitol, urea, formic acid, lactic acid, and methylamine were purchased as 14 C-labeled stock solutions from the American Radiolabeled Company (St. Louis, MO). Atenolol, EGTA, and PC were purchased from Sigma Chemical Company (St. Louis, MO). Hanks' balanced salt solution (HBSS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (HEPES), nonessential amino acids, trypsin/EDTA, and Dulbecco's Modified Eagle Medium (DMEM) were purchased from JRH Biosciences (Lenexa, KS). Penicillin and streptomycin were purchased as a mixture from Irvine Scientific (Santa Ana, CA). Fetal bovine serum and type I rat tail collagen were purchased from Atlanta Biologicals (Norcross, GA) and Collaborative Research (Lexington, MA), respectively. Cell CulturesThe Caco-2 cell line, which originated from a human adenocarcinoma, 15 was obtained from the American Type Culture Collection (Rockville, MD) and was cultured as described previously by Hildago et al. 10 The cells were grown in 150-cm 2 culture flasks
Journal of pharmacy …, 2002
Peptide drugs are hydrophilic in nature and so their preferred pathway of membrane transport is by the paracellular route, which primarily involves passive diffusion across intercellular pores. The objective of the present study was to investigate the effect of secondary structure on the aqueous diffusion of a model polypeptide, poly(L-lysine), through a microporous membrane. The primary aim was to systematically evaluate the variables (e.g. viscosity and/or hydrodynamic radius) that may contribute to the difference, if any, in the calculated values of the aqueous diffusion coef cient (D aq ) for each conformer of poly(L-lysine). Variations in pH and temperature of the medium were used to induce secondary structural changes in poly(L-lysine). Transport studies were conducted for 3 h at 25 or 37°C using side-by-side diffusion cells. Hydrophilic microporous polyester membranes with a 1-m pore diameter were used to measure the free diffusion of each conformer. The values for the apparent permeability (P app ) and D aq were calculated using standard equations. The viscosity of each conformer solution was determined and the hydrodynamic radius of each conformer was then estimated. At 25°C, both P app and D aq of the -helix conformer were approximately the same as those of the random coil conformer. In contrast, at 37°C, the P app and the D aq of the -sheet conformer were signi cantly (P ! 0.05) less than those of the random coil conformer. At 25°C, the solutions containing primarily either the random coil or the -helix conformers had approximately the same viscosity. On the other hand, at 37°C, the solutions containing the -sheet conformer had a signi cantly (P ! 0.05) higher viscosity than when this conformer was absent. The random coil and the -helix conformers appeared to have comparable sizes, whereas the hydrodynamic radius estimated for the -sheet conformer was signi cantly (P ! 0.05) larger than those for the other two conformers. In summary, changing the secondary structure of poly(L-lysine) from the random coil to the -helix did not affect its P app and intrinsic D aq . On the other hand, appearance of the -sheet conformer signi cantly decreased the values of P app and D aq . The differences appeared to result from the signi cantly higher solution viscosity as well as the extended structure associated with the -sheet conformer of poly(Llysine). This strategy may represent a potential mechanism to sustain the delivery of therapeutic peptide drugs from a controlled drug delivery device.
Chemistry of Materials, 2004
The use of polypeptide-functionalized membranes with known porosity and uniform pore sizes allows a better understanding of the separation characteristics in nanodomains. A stimuli-responsive polypeptide, poly-L-glutamic acid (PLGA), was immobilized on a polycarbonate track-etched (PCTE) membrane. First, PCTE membrane was gold-coated under convective conditions, and a thiol, 3-mercapto 1,2-propanediol (MPD), was chemisorbed on the modified surface. Second, the MPD molecule was oxidized with sodium periodate to obtain an aldehyde functionality, which was further reacted with the amino group on the PLGA molecule, making possible a single point polypeptide attachment. The morphology of the modified membrane was analyzed by electron microscopy (SEM, TEM) imaging, confirming uniform structure. Modified membrane performances, with starting diameters of 30 and 100 nm, were evaluated in terms of solute (ionic and neutral) rejections and water permeability. Both solute and water transport through membrane were reversibly regulated by pH. The effective membrane charge was calculated using the extended Nernst-Plank equation coupled with Donnan equilibrium and electroneutrality conditions.
TOPICAL REVIEW Dynamics of polynucleotide transport through nanometre-scale pores
2003
Online at stacks.iop.org/JPhysCM/15/R581 The transport of biopolymers through large membrane channels is a ubiquitous process in biology. It is central to processes such as gene transfer by transduction and RNA transport through nuclear pore complexes. The transport of polymers through nanoscopic channels is also of interest to physicists and chemists studying the effects of steric, hydrodynamic, and electrostatic interactions between polymers and confining walls. Single-channel ion current measurements have been recently used to study the transport of biopolymers, and in particular single-stranded DNA and RNA molecules, through nanometre-size channels. Under the influence of an electric field, the negatively charged polynucleotides can be captured and drawn through the channel in a process termed ‘translocation’. During translocation, the ion current flowing through the channel is mostly blocked, indicating the presence of the polymer inside the channel. The current blockades were ...
Physicochemical Characterization of Transport in Nanosized Membrane Structures
Chemphyschem, 2010
The understanding of polymer–solvent interactions is highly important for the development of tailored membrane manufacturing procedures and for the prediction of membrane performance from transport mechanisms. This study examines the permeation performance of organic solvents through state-of-the-art polyimide membranes (STARMEM, Membrane Extraction Technology Ltd.). Solvents are systematically selected to allow investigation of the effects of key physicochemical transport parameters by keeping constant all other parameters thought to be relevant. The effect of the solubility parameter, polarity (dielectric constant), surface tension, and viscosity are studied in detail. Dead-end permeation experiments are carried out at 20 bar with STARMEM 122 and STARMEM 240 membranes. Results for the selected solvents show higher permeation rates for ketones over alcohols and aromatics as well as for acids. It is suggested that the viscosity and polarity have a greater influence than the other parameters. The effect of solvent molar volume is also investigated. Transport of solvents with high molar volume, independent of their polarity and compatibility with the membrane material, is slower in all cases than for solvents with smaller molar volume. The solubility parameter does not show any significant effect on transport phenomena.
Hindered diffusion of dextran and polyethylene glycol in porous membranes
AIChE Journal, 2000
The effecti®e diffusion coefficients of dextran and polyethylene glycol in track-etched polycarbonate membranes were measured using membranes with nominal pore sizes of 0.03 m, 0.05 m, and 0.1 m. Experiments were performed using narrow-size range fractions of each polymer. When the Stokes-Einstein radius was used to describe solute size, the obser®ed diffusi®ities for both polymers agreed closely and were larger than ®alues predicted for rigid spheres, as well as for linear polymers when only steric interactions with the pore wall are assumed. These obser®ations cannot be explained by considering electrostatic interactions between the solute and the pore wall, or solute adsorption on the pore wall. The experimentally measured diffusion coefficients agreed well with a model that treats the polymeric solutes as rigid spheres and includes ®an der Waals attracti®e interactions between the solute and the pore wall.
Macromolecules, 2009
This work describes proton transport in membranes cast from dimethyl sulfoxide solutions of polyelectrolytes obtained by polycondensation of 4,4 0-diaminodiphenyl ether (ODA) and 4,4 0-diaminodi-phenyl ether-2,2 0-disulfonic acid (ODADS) with 1,4,5,8-naphthalenetetracarboxylic dianhydride (NTDA), the moles of sulfonated diamine per mole of unsulfonated one being roughly 3/1. Pulsed field gradient (PFG) NMR studies reveal two kinds of water: water located in the pores of the membranes appearing in the range 5 to 1 ppm and a minor amount of water associated with the imide groups, appearing at 1 ppm. The diffusion coefficient of 1 H in the first type of water is about 2 orders of magnitude higher than that measured in the second type and in both cases the values of this parameter severely decrease as the water content of the membranes decreases. The diffusion coefficients of bare protons, hydronium ions and water in the membranes were calculated using molecular dynamics techniques. For membranes with low water content, the diffusion coefficient of 1 H is very close to the diffusion coefficients of water and hydronium ions obtained by simulation. At high concentrations the simulated values are higher than D(1 H). The simulated values obtained for the diffusion coefficients of hydronium ion and water for membranes equilibrated with water are fairly close to those estimated, respectively, from proton conductivity and osmotic measurements. This work suggests that the study of cationexchange membranes in the acidic form using NMR, conductivity, and molecular dynamics simulation techniques provides useful information on how structure and water content affect transport processes in membranes.