CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED PHOTOHEMOLYSIS*

Abstract— Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3ß-hydroxy-5α-hydroperoxy-△⁶-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.     

PROTOPORPHYRIN-SENSITIZED PHOTODYNAMIC MODIFICATION OF PROTEINS IN ISOLATED HUMAN RED BLOOD CELL MEMBRANES

Abstract— The photodynamic action of protoporphyrin on red cell ghosts is reflected by extensive cross-linking of membrane proteins to very high molecular weight protein aggregates. This process was studied with sepharose gel chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Most sensitive to this photodynamic effect are spectrin and band 2. 1, 2. 2, 2.3 and 4.1. polypeptides, which are cross-linked after very brief illumination periods, with a concomitant loss of spectrin-associated ATPase activity. Band 6 protein, representing the monomeric form of glyceraldehyde-3-phosphate dehydrogenase, is also very sensitive to protoporphyrin-induced cross-linking. The enzymatic activity decreased even faster than the amount of band 6 polypeptides, suggesting that modification(s) of the enzyme other than cross-linking, possibly by rapid photooxidation of a thiol group, may be responsible for inactivation.
Extracted and purified spectrin was cross-linked with about the same velocity as membrane-bound spectrin, reinforcing our previously drawn conclusion that membrane lipids are not involved in the cross-linking reaction. Eluted band 6 polypeptides on the other hand exhibited a relatively fast photo-oxidative modification but a much slower cross-linking to dimers and tetramers. This suggests that the membrane structure, e.g. the spectrin matrix may play an essential role in the incorporation of membrane-bound band 6 polypeptides in the high molecular weight cross-linked complex.     

Comparative studies on performance of radiation-induced and thermal cross-linked ion-exchange membrane for water electrolysis

Radiation-induced and thermal cross-linked sulfonated poly(ether sulfone) (SPS)–sulfonated poly(ether ether ketone) (SPK) composite ion-exchange membranes (SPS/SPK(γ) and SPS/SPK(T), respectively) were prepared. Their performances for water electrolysis were comparatively assessed. Thermal cross-linked membrane (SPS/SPK(T)) showed cross-linking of part functional groups (–SO₃H) and thus deterioration in membrane conductivity. While, radiation-induced cross-linked membrane (SPS/SPK(γ)) avoided any cross-linking between functional groups and thus conductivity. Electrolysis performances of these membranes were evaluated in comparison with Nafion117 membrane. Relatively low current efficiency (CE) for SPS/SPK and SPS/SPK(T) membranes was due to their high mass transfer (water) via electro-osmotic drag, which was negligible for SPS/SPK(γ) membrane. SPS/SPK(γ) membrane exhibited comparable stabilities and water splitting performance with Nafion117 membrane, which revealed its suitability as substitute for electrochemical applications.     

Preparation and properties of covalently cross-linked sulfonated copolyimide membranes containing benzimidazole groups

A series of sulfonated copolyimides containing benzimidazole groups (SPIs) were synthesized by random copolymerization of 1,4,5,8-naphthalenetetracarboxylic dianhydride (NTDA), 2-(4-aminophenyl)-5-aminobenzimidazole (APABI), 4,4′-diaminodiphenyl ether-2,2′-disulfonic acid (ODADS) and 9,9-bis(4-aminophenyl)fluorene (BAPF) in m-cresol in the presence of benzoic acid and triethylamine at 180 °C for 20 h. Membranes with good mechanical properties were prepared by solution cast method. Proton exchange treatment resulted in ionic cross-linking and the membranes were further covalently cross-linked by treating them in polyphosphoric acid (PPA) at 180 °C for 6 h. The covalently cross-linked membranes displayed slightly lower ion exchange capacities (IECs) and proton conductivities than the corresponding covalently uncross-linked ones because small part of the sulfonic acid groups had been consumed during the cross-linking process. Fenton’s test (3% H₂O₂ + 3 ppm FeSO₄, 80 °C) revealed that benzimidazole groups played an important role in the radical oxidative stability of the membranes, while the cooperative effect of benzimidazole groups and covalent cross-linking led to much more significant enhancements in the radical oxidative stability of the membranes than each alone. The membrane 4 (ODADS/APABI/BAPF = 2/1/1, by mol), for example, after covalent cross-linking could maintain membrane form within 8 h measurement, which was much longer than that (3 h) before covalent cross-linking under the same conditions. The membrane 5 (ODADS/BAPF = 3/1, by mol) without benzimidazole groups, however, after covalent cross-linking started to break into pieces after 85 min measurement, which was only slightly longer than that (60 min) before cross-linking under the same conditions.     

壳聚糖季铵盐/滤纸复合膜对胆红素的吸附研究

用2,3-环氧丙基三甲基氯化铵对壳聚糖进行化学修饰,在壳聚糖的分子结构中引入季胺盐基团,提高其阳离子含量。将壳聚糖季铵盐涂在滤纸上,用戊二醛交联,制得壳聚糖季铵盐/滤纸复合膜,考察该复合膜的强度以及对胆红素的吸附性能。实验结果表明,壳聚糖季铵盐/滤纸复合膜具有良好的力学性能;对胆红素的吸附在3h基本达到平衡,其吸附量远大于壳聚糖/滤纸复合膜;适当取代度及高交联度的复合膜吸附效果较好。复合膜对胆红素的吸附量随离子强度的增加而降低;血清白蛋白的加入使吸附量下降。     

Preparation and characterization of N,O-carboxymethyl chitosan (NOCC)/polysulfone (PS) composite nanofiltration membranes

N,O-carboxymethyl chitosan (NOCC) composite nanofiltration membranes having a polysulfone (PS) UF membrane as the substrate were prepared using a method of coating and cross-linking, in which a glutaraldehyde (GA) aqueous solution was used as the cross-linking agent. Attenuated total reflection infrared spectroscopy (ATR-IR) was employed to characterize the resulting membrane. The effects of the composition of the casting solution of the active layer, the concentration of the cross-linking agent, and the membrane preparation techniques on the performance of the composite membrane were investigated. At 13–15 °C and 0.40 MPa the rejections of the resulting membrane to Na₂SO₄ and NaCl solutions (1000 mg L⁻¹) were 92.7 and 30.2%, respectively, and the permeate fluxes were 3.0 and 5.1 kg m⁻² h⁻¹, respectively. The rejection of this kind of membrane to the electrolyte solutions decreased in the order of Na₂SO₄, NaCl, MgSO₄, and MgCl₂. This suggests that the membrane active layer acquires a negative surface charge distribution by the adsorption of anions from the electrolyte solution and this charge distribution mainly determines the membrane performance.     

Surface modification of nanofiltration membranes to improve the removal of organic micro-pollutants (EDCs and PhACs) in drinking water treatment: Graft polymerization and cross-linking followed by functional group substitution

A commercially available thin film composite (TFC) polyamide (PA) nanofiltration (NF) membrane was chemically modified to improve its rejection capacity for selected organic micro-pollutants categorized as endocrine disrupting chemicals (EDCs) and pharmaceutically active compounds (PhACs): bisphenol-A (BPA), ibuprofen, and salicylic acid. The raw NF membrane was altered using the following modification sequence: graft polymerization (methacrylic acid (MA)-membrane); cross-linking of grafted polymer chains (ethylene diamine (ED)-membrane); and, substitution of functional groups (succinic acid (SA)-membrane). Attenuated total reflective Fourier transform infrared (ATR-FTIR) was used to verify each modification in the sequence: the formation of amide bonds; graft polymerization and cross-linking; and, increased carboxylic acids on the modified membrane. Based on zeta-potential and contact angle measurements, graft polymerization increased the negative charge and hydrophilicity of the raw membrane, while cross-linking replaced carboxylic acid with amide bonds, which made the modified membrane almost neutral at pH 6.5. The water fluxes of the ED- and SA-membranes were similar to that of the raw membrane; however, the water flux of the MA-membranes varied with polymerization time (the membrane polymerized for 15 min revealed ≥20% higher flux than the raw membrane). BPA rejection by the raw membrane was substantially improved from 74% to ≥95% after this series of modifications. However, the rejection capacity of the ED-membrane for ibuprofen and salicylic acid was slightly reduced compared with those of the MA-membrane, which was polymerized for 15 min, due to the lack of an electrical repulsion mechanism. The SA-membrane recovered its negative surface charge and showed a clear enhancement in the rejection of all pollutants.     

交联嵌段磺化聚芳醚砜质子交换膜的制备及性能

为提高磺化聚芳醚砜(SPAES)质子交换膜的质子传导率及稳定性, 制备了一系列交联嵌段SPAES质子交换膜(cbSPAES). 采用嵌段共聚方法, 在P₂O₅存在下, 利用磺酸基团与聚合物主链上活泼氢的脱水反应进行交联改性合成嵌段聚合物. 采用电化学阻抗谱技术测定了cbSPAES膜的质子传导率, 通过测试水中膜平面及厚度方向的尺寸变化率评价膜的尺寸稳定性, 通过加速老化试验评价膜的水解稳定性. 结果表明, 与未交联膜相比, cbSPAES膜的尺寸稳定性及水解稳定性明显提高; 在交联程度相同时, cbSPAES膜的吸水率和质子传导率随着磺化链段长度的增加呈上升的趋势. 如cbSPAES(30/10)-10膜在60 ℃水中的吸水率为65%, 平面方向和厚度方向的尺寸变化率分别为0.16和0.18, 质子传导率达到163 mS/cm.     

Effects of low molecular weight photosensitizer and UV irradiation on gas permeability and selectivity of polyimide membrane

A polyimide membrane, 6FDA–TMPD, was prepared from 5,5′-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]bis-1,3-isobenzofurandione (6FDA), and 2,4,6-trimethyl-1,3-phenylenediamine (TMPD). The effect of ultraviolet irradiation (UV irradiation) using a high-pressure mercury lamp on the gas transport properties of the 6FDA–TMPD membrane and membranes containing a photosensitizer was explored. Comparison between UV treatment and thermal treatment was also examined. The gas selectivities in all membranes were increased due to the irradiation. Especially in the H₂/N₂ and H₂/CH₄ separations, the selectivities effectively increased without a significant reduction in the H₂ permeability. These increases were considerable in the membrane having no photosensitizer which was expected to be an initiator for the cross-linking reaction. There was little effect of cross-linking, which was formed by the irradiation, on the gas permeation properties. Photosensitizers prevented drastic change in the membrane morphologies due to the irradiation.     

Characterization of the mechanical properties of cross-linked serum albumin microcapsules: effect of size and protein concentration

A microfluidic technique is used to characterize the mechanical behavior of capsules that are produced in a two-step process: first, an emulsification step to form droplets, followed by a cross-linking step to encapsulate the droplets within a thin membrane composed of cross-linked proteins. The objective is to study the influence of the capsule size and protein concentration on the membrane mechanical properties. The microcapsules are fabricated by cross-linking of human serum albumin (HSA) with concentrations from 15 to 35 % (w/v). A wide range of capsule radii (～40–450 μm) is obtained by varying the stirring speed in the emulsification step. For each stirring speed, a low threshold value in protein concentration is found, below which no coherent capsules could be produced. The smaller the stirring speed, the lower the concentration can be. Increasing the concentration from the threshold value and considering capsules of a given size, we show that the surface shear modulus of the membrane increases with the concentration following a sigmoidal curve. The increase in mechanical resistance reveals a higher degree of cross-linking in the membrane. Varying the stirring speed, we find that the surface shear modulus strongly increases with the capsule radius: its increase is two orders of magnitude larger than the increase in size for the capsules under consideration. It demonstrates that the cross-linking reaction is a function of the emulsion size distribution and that capsules produced in batch through emulsification processes inherently have a distribution in mechanical resistance.     

Potentiometric phosphate-sensing system utilizing phosphate-binding protein

A phosphate-detection system has been developed which uses phosphate-binding protein (PBP) from Escherichia coli. PBP was immobilized on a sheet of nitrocellulose membrane by cross-linking and the membrane potential of the immobilized PBP was measured. The response time of the system to phosphate was 5 min. The response was selective to phosphate among other anions. Under optimum conditions 0.1-1.5 mmol L(-1) phosphate can be determined with this system.     

Effect of modification of a sulfonic cation-exchange membrane with cross-linked chitosan on the selectivity of the transport of magnesium and calcium ions relative to sodium ions

A procedure was suggested for modification of MK-40 sulfonic cation-exchange membrane by application of a chitosan layer onto its surface, followed by cross-linking of this layer with epichlorohydrin. The transport of magnesium, calcium, and sodium ions through cation-exchange membranes modified with cross-linked and non-cross-linked chitosan was studied.     

Composite hybrid membrane of chitosan-silica in pervaporation separation of MeOH/DMC mixtures

Chitosan-silica hybrid membranes (CSHMs) were prepared by cross-linking chitosan (CS) with 3-aminopropyl-triethoxysilane (APTEOS). The dynamic behaviors of the CS membrane and the CSHM were investigated in pervaporation (PV) of methanol/dimethyl carbonate (MeOH/DMC) mixtures. The membranes were characterized by X-ray diffraction (XRD), contact angle meter, scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The transition state of PV processes were studied. During the PV processes, the amorphous region of the membranes increases and the contact angle between MeOH and the membrane decreases within a range of operating time and then remains almost constant implying a reconstruction occurred on the membrane surface. The silica is well distributed in the CSHM matrix and the thermal stability of the CSHM is enhanced. The time for a PV process to reach a steady state decreases with increasing MeOH concentration or feed temperature, and it is longer for the CSHM than the CS membrane under the same operating condition. Swelling experiments show that the degree of swelling (DS) is greatly depressed by cross-linking CS with APTEOS. Sorption data indicate that the selectivity of solubility and diffusion of the CSHM are greatly improved over the CS membrane. The CSHM presents superior separation behaviors over other membranes with a flux of 1265 g/(hm(2)) and separation factor of 30.1 in PV separation of 70 wt% MeOH in feed at 50 degrees C.     

PERVAPORATION PROPERTIES OF PDMS MEMBRANES CURED WITH DIFFERENT CROSS-LINKING REAGENTS FOR ETHANOL CONCENTRATION FROM AQUEOUS SOLUTIONS

Ethanol perm-selective PDMS/PVDF composite membranes were prepared by curing polydimethylsiloxane (PDMS) with various cross-linking reagents,such as tetraethoxylsilane(TEOS),γ-aminopropyltriethoxylsilane(APTEOS), phenyltrimethoxylsilane(PTMOS) and octyltrimethoxylsilane(OTMOS) as well.The cross-linking density and surface properties of the PDMS active layer were adjusted by varying cross-linking reagents.The pervaporation performance of PDMS membranes cured with different cross-linking reagents was inves...     

Membrane receptor probes: solid-phase synthesis of biotin-Asp-PEG-arvanil derivatives

[structure: see text] A modular, flexible solid-phase synthetic route for the preparation of biotinylated cross-linking probes of membrane receptors is described. The route utilizes an orthogonal protection strategy employing a Pd[0] cleavable allyl linker attached to the probe via an aspartate residue. The versatility of the method is illustrated through the synthesis of a number of arvanil-derived cannabinoid receptor ligands displaying either a photoaffinity or a chemical cross-linking group.     

Acetylcholinesterase Immobilization on Polyacrylamide/Functionalized Multi-walled Carbon Nanotube Nanocomposite Nanofibrous Membrane

In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers’ morphology and conductivity) and chemical (enzyme entrapment) methods.

The effects of transition metal complexes on the permeation of small gas molecules through cellulose acetate membranes

Cellulose acetate (CA) membranes containing RuCl₃·3H₂O and RhCl [P(C₆H₅)₃]₃ were prepared reproducibly. Such membranes, on treatment with CO, formed metal-carbonyl species at relatively low temperature. The Ru-carbonyls formed in CA were quite stable at 40°C in comparison with the Rh-carbonyl species and, interestingly, there was no permeation of CO gas through the ruthenium-containing CA membrane at 40°C. However, the permeation of other gas molecules, such as H₂, N₂ and O₂, through the same membrane was reduced only slightly, probably due to the cross-linking effect of the transition metal complexes in CA. It was found that essentially pure H₂ gas could be recovered from a 1: 1 mixture of H₂ and CO gases using ruthenium-containing cellulose acetate membranes.     

Optical Urea Biosensor Based On Ammonium Ion Selective Membrane

ABSTRACT

An optical urea biosensor was developed by immobilizing an urease enzyme layer on a thin ammonium-selective polymer membrane. The ammonium optical membrane utilized dichlorofluorescein octadecyl ester (DCFOE) as anionic chromophore and nonactin as neutral ionophore. The urease layer was coated on the top of the ammonium layer by gelatin entrapment combined with glutaradehyde cross-linking. Hydrolysis of urea catalyzed by urease produced ammonium ion, which was extracted into the-polymer film to form complexes with nonactin. A proton was released which resulted in a color change of the optical membrane due to charge neutrality principle. The biosensor     

Photoinactivation of Sindbis Virus Infectivity Without Inhibition of Membrane Fusion

Photoinactivation of enveloped viruses is commonly associated with damage to fusion proteins and inhibition of membrane fusion capacity. Here we show that photobleaching of Sindbis virus labeled with the membrane localized dye, R18 (octadecyl rhodamine B) causes a dramatic loss of infectivity without observable changes in low-pH triggered membrane fusion to liposomes. Sindbis labeled with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) also maintains low-pH triggered membrane fusion capacity, but in contrast to R18, extensive photobleaching of DiI-labeled virus has little effect on infectivity. Electrophoretic gel analysis suggests no cross-linking of viral fusion proteins following photobleaching of dye-labeled Sindbis. These observations have implications for live-cell, single particle tracking studies of dye-labeled Sindbis virus. Our observations suggest that R18 and DiI have different propensities for spontaneous flip-flop in lipid bilayers.