Effect of protein adsorption on the transport characteristics of asymmetric ultrafiltration membranes.

The effects of bovine serum albumin adsorption on the transport characteristics of asymmetric poly(ether sulfone) ultrafiltration membranes were determined using polydisperse dextrans with gel permeation chromatography. Actual dextran sieving coefficients were evaluated from observed sieving data for both the clean and preadsorbed membranes using a stagnant film model. The flux dependence of the actual dextran sieving coefficients was used to evaluate the intrinsic membrane hindrance factors for convective (i.e., sieving) and diffusive transport for the different molecular weight dextrans using classical membrane transport theory. Protein adsorption caused a reduction in both dextran sieving and diffusion, with the magnitude of the reduction a function of the dextran molecular weight and pore size. The effects of adsorption on the specific pore area and the membrane porosity were then determined using a recent model for solute transport through asymmetric ultrafiltration membranes. The data indicate that protein adsorption occurs preferentially in the larger membrane pores, causing a greater reduction in solute sieving compared to the membrane hydraulic permeability and porosity than would be predicted on the basis of either a simple pore blockage or pore constriction model.     

A finite deformation theory for cartilage and other soft hydrated connective tissues--I. Equilibrium results

The determination of valid stress-strain relations for articular cartilage under finite deformation conditions is a prerequisite for constructing models for synovial joint lubrication. Under physiological conditions of high strain rates and/or high stresses in the joint, large strains occur in cartilage. A finite deformation theory valid for describing cartilage, as well as other soft hydrated connective tissues under large loads, has been developed. This theory is based on the choice of a specific Helmholtz energy function which satisfies the generalized Coleman-Noll (GCN0) condition and the Baker-Ericksen (B-E) inequalities established in finite elasticity theory. In addition, the finite deformation biphasic theory includes the effects of strain-dependent porosity and permeability. These nonlinear effects are essential for properly describing the biomechanical behavior of articular cartilage, even when strain rates are low and strains are infinitesimal. The finite deformation theory describes the large strain behavior of cartilage observed in one-dimensional confined compression experiments at equilibrium, and it reduces to the linear biphasic theory under infinitesimal strain and slow strain rate conditions. Using this theory, we have determined the material coefficients of both human and bovine articular cartilages under large strain conditions at equilibrium. The theory compares very well with experimental results.     

Partitioning and diffusion of proteins and linear polymers in polyacrylamide gels.

The equilibrium partition coefficient (K) and diffusion coefficient (Dgel) of two proteins and two linear polymers were measured as a function of polymer content of a 2.7% cross-linked polyacrylamide (PA) gel. The gel concentration, expressed as a volume percentage of PA in the gel (phi), varied between 0 and 14%. The measurements were made by fluorescence spectroscopy; fluorescent dyes were covalently attached to the macromolecules. The dependence of K on phi for the proteins agrees with a model of the gel network as randomly placed, impenetrable rods. The diffusion data are interpreted in terms of an effective medium theory for the mobility of a sphere in a Brinkman fluid. Using values of the Brinkman parameter in the literature, the effective medium model with no adjustable parameters fits the diffusion data for the proteins very well but underpredicts Dgel for the linear polymers. The gel effect on partitioning is significantly greater than that on diffusion. The permeability (KDgel) of bovine serum albumin decreased by 10(3) over the range phi = 0 --> 8%, and the ratio of permeabilities for ribonuclease compared to BSA increased from 2 to 30.     

The internal mechanics of the intervertebral disc under cyclic loading

The mechanics of the intervertebral disc (IVD) under cyclic loading are investigated via a one-dimensional poroelastic model and experiment. The poroelastic model, based on that of Biot (J. Appl. Phys. 12 (1941) 155; J. Appl. Mech. 23 (1956) 91), includes a power-law relation between porosity and permeability, and a linear relation between the osmotic potential and solidity. The model was fitted to experimental data of the unconfined IVD undergoing 5 cyclic loads of 20 min compression by an applied stress of 1MPa, followed by 40 min expansion. To obtain a good agreement between experiment and theory, the initial elastic deformation of the IVD, possibly associated with the bulging of the IVD into the vertebral bodies or laterally, was removed from the experimental data. Many combinations of the permeability-porosity relationship with the initial osmotic potential (pi(i)) were investigated, and the best-fit parameters for the aggregate modulus (H(A)) and initial permeability (k(i)) were determined. The values of H(A) and k(i) were compared to literature values, and agreed well especially in the context of the adopted high-stress testing regime, and the strain related permeability in the model.     

枯草芽孢杆菌中性β-甘露聚糖酶的纯化及性质研究

经硫酸铵分级沉淀、超滤浓缩、阴离子交换层析和凝胶过滤层析,由枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）ＢＭ９６０２培养滤液得到了常规凝胶电泳一条带的中性β－甘露聚糖酶．该酶具有与其它已知同类酶相类似的性质,但用ＳＤＳ－ＰＡＧＥ测得该酶分子量为３７ｋＤ;用聚丙烯酰胺等电聚焦电泳测得等电点ｐＩ为４．９．     

Hindered diffusion in agarose gels: test of effective medium model.

The diffusivities of uncharged macromolecules in gels (D) are typically lower than in free solution (D infinity), because of a combination of hydrodynamic and steric factors. To examine these factors, we measured D and D infinity for dilute solutions of several fluorescein-labeled macromolecules, using an image-based fluorescence recovery after photobleaching technique. Test macromolecules with Stokes-Einstein radii (rs) of 2.1-6.2 nm, including three globular proteins (bovine serum albumin, ovalbumin, lactalbumin) and four narrow fractions of Ficoll, were studied in agarose gels with agarose volume fractions (phi) of 0.038-0.073. The gels were characterized by measuring the hydraulic permeability of supported agarose membranes, allowing calculation of the Darcy permeability (kappa) for each gel sample. It was found that kappa, which is a measure of the intrinsic hydraulic conductance of the gel, decreased by an order of magnitude as phi was increased over the range indicated. The diffusivity ratio D/D infinity, which varied from 0.20 to 0.63, decreased with increases in rs or phi. Thus as expected, diffusional hindrances were the most severe for large macromolecules and/or relatively concentrated gels. According to a recently proposed theory for hindered diffusion through fibrous media, the diffusivity ratio is given by the product of a hydrodynamic factor (F) and a steric factor (S). The functional form is D/D infinity = F(rs/k1/2) S(f), where f = [(rs+rf)/rf]2 phi and rf is the fiber radius. Values of D/D infinity calculated from this effective medium theory, without use of adjustable parameters, were in much better agreement with the measured values than were predictions based on other approaches. The strengths and limitations of the effective medium theory for predicting diffusivities in gels are discussed.     

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.     

Localization of a sheep pineal antigonadotropin

An antigonadotropic substance was partially purified from aqueous extracts of sheep pineal bodies by gel filtration on Sephadex G-25 and ultrafiltration using the Amicon Diaflo membranes UM-2 and UM-05. When injected into male and female Charles River CD-1 albino mice the low molecular weight fractions from Sephadex G-25, and after ultrafiltration the UM-05 residue of these fractions, demonstrated significant effects on the reproductive system. These effects included the inhibition of compensatory ovarian hypertrophy in unilaterally ovariectomized adult mice, the reduction of ventral prostate weights in adult mice, and the reduction of gonad and accessory reproductive organ weights in young male and female mice.Further purification of the antigonadotropic substance was accomplished by gel filtration of the UM-05 residue on Sephadex G-10. The elution volume of the biologically active fraction did not correspond to that of synthetic melatonin applied subsequently to the same column. The antigonadotropic activity in the UM-05 residue was localized by paper electrophoresis and paper chromatography.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Analysis of protein fouling during ultrafiltration using a two-layer membrane model

Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport.     

Conductance of porous media depends on external electric fields

In obstacle-filled media, such as extracellular or intracellular lumen of brain tissue, effective ion-diffusion permeability is a key determinant of electrogenic reactions. Although this diffusion permeability is thought to depend entirely on structural features of the medium, such as porosity and tortuosity, brain tissue shows prominent nonohmic properties, the origins of which remain poorly understood. Here, we explore Monte Carlo simulations of ion diffusion in a space filled with overlapping spheres to predict that diffusion permeability of such media decreases with stronger external electric fields. This dependence increases with lower medium porosity while decreasing with radial (two-dimensional or three-dimensional) compared with homogenous (one-dimensional) fields. We test our predictions empirically in an electrolyte chamber filled with microscopic glass spheres and find good correspondence with our predictions. A theoretical insight relates this phenomenon to a disproportionately increased dwell time of diffusing ions at potential barriers (or traps) representing geometric obstacles when the field strength increases. The dependence of medium ion-diffusion permeability on electric field could be important for understanding conductivity properties of porous materials, in particular for the accurate interpretation of electric activity recordings in brain tissue.     

Biomechanical properties of human articular cartilage under compressive loads

The function of articular cartilage is to support and distribute loads and to provide lubrication in the diarthrodial joints. Cartilage function is described by proper mechanical and rheological properties, strain and depth-dependent, which are not completely assessed. Unconfined and confined compression are commonly used to evaluate the Young's modulus (E) and the aggregate modulus (H(A)), respectively. The Poisson's ratio (nu) can be calculated indirectly from the equilibrium compression data, or using the biphasic indentation technique; it has recently been optically evaluated by using video microscopy during unconfined compression. The transient response of articular cartilage during confined compression depends on its permeability k; a constant value of k can be easily identified by a simple analytical model of confined compression tests, whereas more complex models or direct measurements (permeation tests) are needed to study the permeability dependence on deformation. A poroelastic finite element model of articular cartilage was developed for this purpose. The elastic parameters (E,nu) of the model were evaluated performing unconfined compression creep tests on human articular cartilage disks, whereas k was identified from the confined test response. Our combined experimental and computational method can be used to identify the parameters that define the permeability dependence on deformation, as a function of depth from articular surface.     

Identification and Purification of an External Antigenic Glycoprotein (Immobilization Antigen) From Pseudomicrothorax Dubius

SYNOPSIS. A large, external glycoprotein with antigenic properties isolated from the ciliate Pseudomicrothorax dubius was found to have a molecular weight of ∼ 250,000 daltons. Analysis of the extracts by isoelectric focusing in combination with immunodiffusion and gradient polyacrylamide gel electrophoresis revealed that the principal antigen was a large glycoprotein. the glycoprotein was purified partially by Sephadex ultrafiltration. and almost completely by affinity chromatography on a concanavalin A-Sepharose column.     

Binding of radioiodinated human beta-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).     

A fiber matrix model for interstitial fluid flow and permeability in ligaments and tendons

Collagen fibrils in ligaments and tendons are highly organized into parallel arrays which influence interstitial fluid transport. Finite element (FE) models were developed analogous to the fibrillar arrays in ligaments and tendons to investigate interstitial fluid flow and tissue permeability as a function of interfibrillar spacing and fluid properties. Collagen fibrils were assumed to be a periodic square array of impermeable cylinders. A two-dimensional FE model was used to study transverse fluid flow and a three-dimensional model was used to study flow parallel to the collagen fibrils. Parametric FE analysis provided data to formulate empirical expressions for permeability (kappa) as a function of porosity (phi). Results show that longitudinal permeability (kappa = 1.1.10(-15)phi 2.5[1 - phi]-0.333) can be up to 50 times higher than transverse permeability (kappa = 1.2.10(-15)phi 0.5[phi - phi min]2.5) in a compact array. Maximum fluid shear stresses occur at the narrowest zones of adjacent fibrils (1.21 Pa or 12.1 dyn/cm2 at 10 microns/s of average transverse influx). If interstitial fluid is highly non-Newtonian, the permeability should be considered as flow (shear)-dependent. The computational results suggest that tissue permeability in ligaments and tendons is highly anisotropic, porosity-dependent, and can be estimated by analytic expressions.     

Purification and characterization of a thermostable xylanase from Fomitopsis pinicola

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular weight of F. pinicola xylanase was determined to be 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of 70 degreesC. The enzyme showed t(1/2) value of 33 h at 70 degrees C and catalytic efficiency (k(cat) = 77.4 s?1, k(cat)/K(m) = 22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.     

Filtration,diffusion, and molecular sieving through porous cellulose membranes

1. A study has been made of the diffusion and filtration of a graded series of molecules (including tritium-labelled water, urea, glucose, antipyrine, sucrose, raffinose, and hemoglobin) in aqueous solution through porous cellulose membranes of three degrees of porosity. 2. Experimental results were in close agreement with predictions based on the membrane pore theory of Pappenheimer et al. (1,2). Restriction to molecular diffusion is a function of pore radius and molecular radius described by equation (11) in the text. Molecular sieving during ultrafiltration is a function of total pore area per unit path length, pore radius, molecular radius, and filtration rate given by equations (16) and (19). 3. Estimates of average pore radius made by means of this theory were considerably larger than estimates made by the method of Elford and Ferry (3) (Table II). Sources of error in the latter method are discussed and a new method of membrane calibration is proposed in which the total cross-sectional area of the pores is measured by direct diffusion of isotope-labelled water. 4. Steady-state osmotic pressures of solutions of sucrose and raffinose measured during molecular sieving through cellulose membranes were found to be close to the "ideal" osmotic pressures calculated by van't Hoff's law. Thus the present experimental data support the methods used by Pappenheimer et al. in their studies on living capillary walls as well as their theory of membrane pore permeability.     

Purification and characterization of xylanase from a thermophilic Streptomyces sp. K37

Extracellular xylanase (EC 3.2.1.8) from Streptomyces sp. K37 was purified 33.53 by ultrafiltration and cation exchange chromatography followed by gel filtration chromatography. The optimum pH and temperature for purified xylanase were found to be pH 6.0 and 60 degrees C. The Km and V(max) values of the purified xylanase were 15.4 mg ml(-1) and 0.67 micromole reducing sugar min(-1) ml(-1). High performance liquid chromatography (HPLC) gel filtration of the purified xylanase eluted xylanase activity as a peak corresponding to the molecular weight of about 24.3 kDa while the molecular weight determined by SDS-PAGE was found to be 26.4 kDa. The purified xylanase of Streptomyces sp. K37 was found to be endoxylanase and non arabinose liberating enzyme and was highly glycosylated (73.97%).     

Isolation and characterization of the K5-type yeast killer protein and its homology with an exo-beta-1,3-glucanase

K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogeneity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinuous gradient SDS-PAGE and had a molecular mass of 49,000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immuno assay revealed that it was a glycosylated protein. Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-beta-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37 degrees C.     

Purification of cytochrome b from complex III of beef heart mitochondria

Two cytochrome b preparations have been prepared from Complex III of beef heart mitochondria, by detergent-exchange chromatography on a butyl-Toyopearl column. One was eluted from the column with buffer containing Tween 20 after most of other subunits of Complex III were eluted with buffer containing guanidine-HCl, and the other was eluted from the column with buffer containing sodium dodecyl sulfate. The former is consisted of a single polypeptide (subunit III) and contained 37.5 nmol of heme b/mg of protein, and the latter consisted of subunits III and IX and contained 19.5 nmol of heme b/mg of protein. The former was labile when it was reduced by dithionite, whereas the latter was stable. Subunit IX in the latter is associated with cytochrome b even after gel filtration and density gradient centrifugation. These results suggest that subunit IX plays a role in stabilizing cytochrome b.