Glucose-responsive polymer bearing a novel phenylborate derivative as a glucose-sensing moiety operating at physiological pH conditions

This study is devoted to the development of novel glucose-responsive polymers that operate under physiological conditions (pH 7.4, 37 degrees C), aiming for future use in a self-regulated insulin delivery system to treat diabetes mellitus. The approach involves the use of a newly synthesized phenylborate derivative [4-(1,6-dioxo-2,5-diaza-7-oxamyl) phenylboronic acid, DDOPBA] possessing an appreciably low pK(a) ( approximately 7.8) as a glucose-sensing moiety, as well as the adoption of poly(N-isopropylmethacrylamide), PNIPMAAm, as the main chain that exhibits critical solution behavior in the range close to physiological temperature. Glucose- and pH-dependent changes in the critical solution behavior of the resultant copolymers were investigated at varying temperatures, revealing definite glucose sensitivities near the physiological conditions. Furthermore, DDOPBA moieties in the copolymers maintained constant apparent pK(a) values even when the temperature approaches the critical solution points of the main chain, indicating that spacing of the phenylborate moiety from the polymer backbone is a feasible way to minimize the microenvironment effect caused by a temperature-induced change in the hydration state of the polymer strands.     

Bio-based hydrogels prepared by cross-linking of microbial poly(gamma-glutamic acid) with various saccharides

Novel bio-based hydrogels were prepared by cross-linking of microbial poly(gamma-glutamic acid) (PGA) with saccharides such as glucose, maltotriose, and cyclodextrin (CD) in the presence of water-soluble carbodiimide in dimethyl sulfoxide (DMSO) by one-pot synthesis at 25 degrees C for 24 h. The degradation of the gels in alkaline solution (pH 9) at 37 degrees C was also investigated. The PGA gels cross-linked with various neutral saccharides were obtained in relatively high recovery yields by use of a base like 4,4-(dimethylamino)pyridine. The PGA gel cross-linked by glucose showed the highest water absorption of 3000 g/g. The PGA gels cross-linked by CDs showed higher water absorption than those cross-linked by the corresponding linear saccharides. It was revealed that the water absorption of the PGA gel was affected by the cross-linker content and also the structure of cross-linkers as they had an effect on the cross-linking density of the PGA gel. The PGA gels were hydrolyzed under alkaline condition (pH 9) at 37 degrees C. The degradation rate was higher when the cross-linker content of the gel was lower.     

Glucose-responsive vehicles containing phenylborate ester for controlled insulin release at neutral pH

This study is devoted to developing amphiphilic block polymers based on phenylborate ester, which can self-assemble to form nanoparticles, as a glucose-sensitive drug carrier. Poly(ethylene glycol)-block-poly[(2-phenylboronic esters-1,3-dioxane-5-ethyl) methylacrylate] (MPEG5000-block-PBDEMA) was fabricated with MPEG5000-Br as a macroinitiator via atom transfer radical polymerization (ATRP). Using the solvent evaporation method, these block polymers can disperse in aqueous milieu to self-assemble into micellar aggregates with a spherical core-shell structure. Zeta potential and fluorescence techniques analysis showed a good purification effect, high encapsulation efficiency, and loading capacity of fluorescein isothiocyanate (FITC)-insulin-loaded polymeric micelles under optimal conditions. The in vitro insulin release profiles revealed definite glucose-responsive behavior of the polymeric micelles at pH 7.4 and 37 °C, depending on the environmental glucose concentration and the chemical composition of the block polymers. Further, circular dichroism spectroscopy demonstrated that the overall tertiary structure of the released insulin was in great agreement with standard insulin. (1)H NMR results of the polymeric micelles during glucose-responsive process supposed one possible insulin release mechanism via the polymer polarity transition from amphiphilic to double hydrophilic. The analysis of L929 mouse fibroblast cells viability suggested that the polymeric micelles from MPEG5000-block-PPBDEMA had low cell toxicity. The block polymers containing phenylborate ester that responded to changes in the glucose concentration at neutral pH are being aimed for use in self-regulated insulin delivery.     

Thin semitransparent gels containing phenylboronic acid: porosity, optical response and permeability for sugars

Radical copolymerization of acrylamide (Am) (90 mol%) with N-acryloyl-m-aminophenylboronic acid (NAAPBA) (10 mol%) carried out on the surface of glass slides in aqueous solution and in the absence of chemical cross-linkers, resulted in the formation of thin semitransparent gels. The phenylboronic acid (PBA) ligand density was ca. 160 micromol/ml gel. The gels exhibited a macroporous structure and displayed optical response to sucrose, lactose, glucose and fructose in 50 mM sodium phosphate buffer, in the pH range from 6.5 to 7.5. The response was fairly reversible and linearly depended on glucose concentration in the wide concentration range from 1 to 60 mM at pH 7.3. The character of response was explained by the balance of two competing equilibrium processes: binding of glucose to phenylboronate anions and binary hydrophobic interactions of neutral PBA groups. The apparent diffusion coefficient of glucose in the gels was ca. 2.5 x 10(-7) cm(2)/s. A freshly prepared gel can be used daily for at least 1 month without changes in sensitivity. Autoclaving (121 degrees C, 1.2 bar, 10 min) allows for the gels sterilization, which is important for their use as glucose sensors in fermentation processes.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Effect of physicochemical parameters on the formation of chitosan-based gels

The possibility for forming physical gels based on Pchelozan (bee chitosan with a molecular weight of 230 kDa and an acetylation degree of 26-65%) has been demonstrated. Conditions for obtaining the gels (1% solution in 1% glycolic acid, 25 degrees C, pH 5.5-7.5) were selected. The effects of (1) the concentration of the original solution, (2) the degree of acetylation of Pchelozan, and (3) the value of pH on the process of gel formation, were studied. The gels obtained may be classified with reversible toxotropic systems. These gels are stable for a long time when stored within a temperature range of 18-55 degrees C. The gel with a degree of acetylation of 34% was characterized by irreversible syneresis.     

Gelatinization and freeze-concentration effects on recrystallization in corn and potato starch gels

Freeze-concentration of starch gels was controlled by temperature and gelatinization with glucose and lactose. The aim of the study was to evaluate the effects of freezing temperature and gel composition on starch recrystallization behaviour of corn and potato starch gels (water content 70%, w/w) in water or glucose or lactose (10%, w/w) solutions. Starch gels were obtained by heating in differential scanning calorimetry (DSC). Samples of starch gels were frozen at -10 degrees C, -20 degrees C and -30 degrees C for 24h and, after thawing, stored at +2 degrees C for 0, 1, 2, 4 and 8 days. The extent of starch recrystallization was taken from the enthalpy of melting of the recrystallized starch by DSC. Freezing temperatures, glucose, lactose and the origin of the starch affected the recrystallization behaviour greatly. The recrystallization of amorphous starch during storage was enhanced by freeze-concentration of gels at temperatures above T'(m). Molecular mobility was enhanced by unfrozen water and consequently molecular rearrangements for nucleation could take place. Further storage at a higher temperature enhanced the growth and the maturation of crystals. In particular, glucose decreased the T'(m) of the gels and consequently lower freezing temperatures were needed to reduce enhanced recrystallization during storage. Freeze-concentration temperatures also showed a significant effect on the size and the perfection of crystals formed in starch recrystallization.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Methacrylated glycol chitosan as a photopolymerizable biomaterial

Glycol chitosan is a derivative of chitosan that is soluble at neutral pH and possesses potentially useful biological properties. With the goal of obtaining biocompatible hydrogels for use as tissue engineering scaffolds or drug delivery depots, glycol chitosan was converted to a photopolymerizable prepolymer through graft methacrylation using glycidyl methacrylate in aqueous media at pH 9. N-Methacrylation was verified by both (1)H NMR and (13)C NMR. The degree of N-methacrylation, measured via (1)H NMR, was easily varied from 1.5% to approximately 25% by varying the molar ratio of glycidyl methacrylate to glycol chitosan and the reaction time. Using a chondrocyte cell line, the N-methacrylated glycol chitosan was found to be noncytotoxic up to a concentration of 1 mg/mL. The prepolymer was cross-linked in solution using UV light and Irgacure 2959 photoinitiator under various conditions to yield gels of low sol content ( approximately 5%), high equilibrium water content (85-95%), and thicknesses of up to 6 mm. Cross-polarization magic-angle spinning (13)C solid state NMR verified the complete conversion of the double bonds in the gel. Chondrocytes seeded directly onto the gel surface, populated the entirety of the gel and remained viable for up to one week. The hydrogels degraded slowly in vitro in the presence of lysozyme at a rate that increased as the cross-link density of the gels decreased.     

Sulfonamide-based pH- and temperature-sensitive biodegradable block copolymer hydrogels

Novel pH- and temperature-sensitive biodegradable poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol) (PCLA-PEG) block copolymers were synthesized with oligomeric sulfamethazine (OSM) end groups (OSM-PCLA-PEG-PCLA-OSM). Aqueous solutions of these block copolymers have shown sol-gel transition behavior upon both temperature and pH changes under physiological conditions (37 degrees C, pH 7.4). The sol-gel transition of these block copolymer solutions was fine-tuned by controlling the PEG length, the hydrophobic to hydrophilic block ratio (PCLA/PEG), and the molecular weight of the sulfamethazine oligomer. Since changes in temperature do not induce gel formation in this pH- and temperature-sensitive block copolymer solution, this hydrogel can be employed as an injectable carrier using a long guide catheter into the body. In addition, the pH of the block copolymer solution showed no change following PCLA degradation over 1 month, and no indication of gel collapse was observed on addition of buffer solution. As such, these properties make the OSM-PCLA-PEG-PCLA-OSM hydrogel an ideal candidate for use as an injectable carrier for certain protein-based drugs known to denature in low-pH environments.     

Purification and properties of a carboxymethylcellulase from phytopathogenic fungus Macrophomina phaseolina

From the culture filtrate of Macrophomina phaseolina, two forms of carboxymethylcellulase were separated by ion-exchange chromatography and designated as CMCase-I and CMCase-II. CMCase-I was purified following a four-step procedure involving gel filtration on Sephadex G-75, Con-A Sepharose 4B affinity chromatography, fast protein liquid chromatography on mono Q anion-exchanger and on Superose 12 gel filtration. The final preparation was homogeneous by SDS-PAGE, isoelectric focussing in thin layers of polyacrylamide gels and immunoelectrophoresis. The enzyme showed optimum activity at pH 5.5 and 65 degrees C, was stable to heating at 65 degrees C for 10 min, and retained 31% of original activity after heating at 80 degrees C for 10 min. The molecular weight of the enzyme was 3.5 x 10(4) Da. A Km of 0.25 mg/ml was determined using carboxymethyl-cellulose as the substrate.     

Gel filtration chromatography of triple-helical calf skin collagen

Gel filtration of type I collagen has been of limited use, because at low pH where the protein is not associated it binds to agarose gels, and at neutrality collagen has a tendency to form fibrils. The more porous polyacrylamide-based gels do not interact with collagen but cannot be used at very high flow rates because they are compressible. It was found that these difficulties are surmounted by use of Fractogel TSK HW-65F, a spherical gel made from a weakly hydrophilic vinyl polymer, and use of the buffer system 0.5 m urea, 0.117 m Tris-HCl, pH 7.3, which prevents fibril formation. The solvent has only a slight effect on the thermal stability of collagen, as determined by circular dichroism measurements. The recovery of native collagen, at 25°C, was at least 88% and that of partially unfolded collagen, at 35°C where it is about one-third unfolded, was 98%. The Fractogel TSK gels and the urea, Tris solvent system should be useful for both preparative work and for studies involving interaction of unaggregated type I collagen with smaller molecules at physiological pH.     

Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization

Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).     

A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.     

Evidence for the glycoprotein nature of retina glycogen

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.     

Purification and characterization of acid phosphatase-1 from Drosophila melanogaster

Acid phosphatase-1 (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2), the major phosphatase in adult Drosophila melanogaster, has been purified to apparent homogeneity. The final product is a glycoprotein homodimer with a subunit molecular weight of about 50,000, as measured by its electrophoretic mobility in denaturing conditions on polyacrylamide gels containing sodium dodecyl sulfate. It has a turnover number of 1720 1-naphthyl phosphate molecules hydrolyzed/s by each acid phosphatase-1 molecule at 37 degrees C, pH 5.0. An average fly contains about 5 ng of enzyme. Pure acid phosphatase-1 displays heterogeneity in isoelectric focusing, with a major band at pH 5.3. The enzyme hydrolyzes a wide variety of phosphate monoesters, including AMP, glucose 6-phosphate, ATP, choline phosphate, or phosphoproteins. The maximum reaction rates are different for all substrates, and some substrates appear to inhibit the reaction at high substrate concentrations. The Michaelis constants for 1-naphthyl phosphate and p-nitrophenyl phosphate are 79 microM and 68 microM, respectively, at pH 5.0 and 37 degrees C. The optimum pH level for 1-naphthyl phosphate is 4.5. Acid phosphatase-1 is inhibited by L(+)-tartrate (but not D(-)-tartrate), phosphate, and fluoride. The reaction rate increases 2.1-fold for every 10 degrees C rise in temperature. Above 48 degrees C, the rate of thermal denaturation is greater than the rate of the enzyme reaction.     

Refolding of the mixed disulfide of bovine trypsinogen and glutathione.

The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Aqueous gel formation of a synthetic peptide derived from the beta-sheet domain of platelet factor-4

We observed gelation of a 23-residue peptide derived from the beta-sheet domain of platelet factor-4 (PF4(24)(-)(46)). The gels were primarily heterogeneous mixtures of 50-200 microm spherical aggregates in a less-dense gel matrix. Infrared and circular dichroism spectroscopies showed gelation involving the conversion of PF4(24)(-)(46) from random coil to beta-sheet. We used aggregation-induced NMR resonance broadening to show that temperature, pH, and ionic strength influenced PF4(24)(-)(46) gelation rates. Under identical solution conditions, gel formation took days at T /= 50 degrees C. Gelation was most rapid at pH values near the pK(a) of the central His35 residue. Increases in solution ionic strength reduced the critical gelation concentration of PF4(24)(-)(46). Our results suggest that PF4(24)(-)(46) gels by a process combining aspects of both heat-set and beta-fibril gelation mechanisms.     

pK-matched running buffers for gel electrophoresis.

Electrophoresis through agarose and polyacrylamide-type gels is the standard method to separate, identify, and purify nucleic acids. Properties of electrophoresis buffers such as pH, ionic strength, and composition affect performance. The buffers in use contain a weak acid or weak base buffered by a compound with a dissimilar pK. Herein, three pK-matched buffers were developed, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25 degrees C (within 0.3 pK units): (i) Ethanolamine/Capso, pH 9.6; (ii) triethanolamine/Tricine, pH 7.9; and (iii) Bis-Tris/Aces, pH 6.7. On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/acetate, pH 8.0) or TBE (Tris/borate, pH 8.3) buffers. The buffers were also tested in 7 M urea denaturing LongRanger sequencing gels and in nondenaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions (up to 200x), and lower current per unit voltage, resulting in less heat generation.