Molecular characteristics of bovine serum albumin-dextran conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5-1 mol of dextran could be bound to 1 mol of native BSA. SDS-PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95-2.35 x 10(5) g/mol and a less-abundant fraction with aggregates greater than 1.50 x 10(6) g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Separation of a series of chromophores and fluorophores present in elastin.

Purified elastin was hydrolysed with HCl and manipulated under conditions that minimized oxidation. Gel-permeation chromatography on polyacrylamide gel and ion-exchange chromatography on dextran cation-exchanger each resulted in the separation of a series of yellow fluorescent fractions. These hitherto unreported ampholytes have fluorescence spectra that approximate to that of the intact protein, and account for its characteristic optical properties. Since the coloured fluorophores are confined to enzyme-resistant regions of the protein molecule they appear to have important structural implications.     

D-Glucosyltransferase of Streptococcus mutans: isolation of two forms of the enzyme that bind to insoluble dextran

The D-glucosyltransferase from Streptococcus mutans 6715 has been separated into three enzymic fractions that differ in their binding to dextran and in their synthesis of dextran from sucrose. One enzymic fraction (AFF-I) does not bind to insoluble dextran, and it produces an insoluble D-glucan. Fraction AFF-IIU was eluted from a dextran affinity-column by either dextran or urea, whereas fraction AFF-IID was eluted only by dextran. Both of these fractions produce insoluble D-glucans from sucrose.     

Molecular Characteristics of Bovine Serum Albumin-Dextran Conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5–1 mol of dextran could be bound to 1 mol of native BSA. SDS–PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95–2.35×10⁵ g/mol and a less-abundant fraction with aggregates greater than 1.50×10⁶ g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Immunoglobulins acquire the ability to interact with DNA after chromatography on QAE-Sephadex

The composition of IgG isolated from normal human serum includes a significant amount of antibodies interacting with native DNA. This ability of antibodies is revealed only after anion-exchange chromatography, resulting in the division of IgG into two fractions, i.e., acid and alkaline immunoglobulins. The peculiarities of interaction of the both fractions with DNA and the specificity of this reaction were investigated. The IgG tested were shown to interact with native and denaturated DNA, dextran sulfate, poly-G and poly-I. The possibility of interaction of antibodies with the charged structures of the cell and their significance under normal and pathological conditions are discussed.     

Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.     

Agarose-dextran gels as synthetic analogs of glomerular basement membrane: water permeability

Novel agarose-dextran hydrogels were synthesized and their suitability as experimental models of glomerular basement membrane was examined by measuring their Darcy (hydraulic) permeabilities (kappa). Immobilization of large dextran molecules in agarose was achieved by electron beam irradiation. Composite gels were made with agarose volume fractions (phi(a)) of 0.04 or 0.08 and dextran volume fractions (phi(d)) ranging from 0 to 0.02 (fiber volume/gel volume), using either of two dextran molecular weights (500 or 2000). At either agarose concentration and for either size of dextran, kappa decreased markedly as the amount of dextran was increased. Statistically significant deviations from the value of kappa for pure agarose were obtained for remarkably small volume fractions of dextran: phi(d) > or = 0.0003 for phi(a) = 0.04 and phi(d) > or = 0.001 for phi(a) = 0.08. The Darcy permeabilities were much more sensitive to phi(d) than to phi(a), and were as much as 26 times smaller than those of pure agarose. Although phi(d) was an important variable, dextran molecular weight was not. The effects of dextran addition on kappa were described fairly well using simple structural idealizations. At high agarose concentrations, the dextran chains behaved as fine fibers interspersed among coarse agarose fibrils, whereas, at low concentrations, the dextran molecules began to resemble spherical obstacles embedded in agarose gels. The ability to achieve physiologically relevant Darcy permeabilities with these materials (as low as 1.6 nm2) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices.     

Molecular sieve chromatography of biosynthetic cell (14C)glucomannan chains

Radioactive d-glucomannan chains, prepared using Phaseolus aureus enzymes, were acetylated and subjected to molecular sieve chromatography. A comparison with dextran acetates, of known molecular weight range, provided approximate molecular weight data. The [¹⁴C]glucomannan chains were not uniformly dispersed, but were separated into two major fractions. These fractions may be collections of polysaccharide chains incompletely resolved. The two fractions had a mobility similar to that of dextrans with molecular weights of 200,000–300,000 and 60,000–90,000. The molecular weight of the largest [¹⁴C]-glucomannan fragment is, consequently, around 200,000 at the minimum. Preliminary results suggest that the lower molecular weight components may be precursors of the higher molecular weight components. Mild acid and alkaline treatment cause the production of materials of much lower molecular size.     

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for "supplementing" properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56 degrees C. for 30 min. destroyed the supplementing activity of each of these fractions. Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C(1)1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C(1)2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C(1)1 by this method.     

Optimizing the conditions of dextran synthesis by the bacterium Leuconostoc mesenteroides grown in a molasses-containing medium

Maximal dextran production (54-55 g/l) by the bacterium Leuconostoc mesenteroides strain V-2317D was observed in molasses-containing media in the presence of 17.5% glucose at pHinit 6.75. The beginning of dextran production depended on the amount of inoculate; maximum yield was observed at a shaker rate of 200 rpm. The dextran produced by L. mesenteroides grown in the molasses-containing medium was representative of three fractions differing in the molecular weight and composition: the high- (approximately 54.5%), medium- (approximately 27.9%), and low-molecular-weight (approximately 2.85%) fractions.     

Affinity chromatography of human anti-dextran antibodies isolation of two distinct populations

Affinity chromatography is a very efficient method for antibody purification. Two affinity chromatography supports were prepared to analyze the specificity of anti-dextran antibodies. Silica beads were grafted with native dextran or with functionalized dextran. The anti-dextran antibodies present in some human sera were analyzed by enzyme-linked immunosorbent assay method. These antibodies play an important role in severe dextran-induced anaphylactic reactions in humans by forming immune complexes with clinical dextran. The results indicated that two distinct populations of anti-dextran antibodies were purified from human serum, using dextran-coated silica beads. Elution from this support with an oligo-dextran of 4000 g/mol allowed the isolation of one population that only recognized native dextran as antigen. Functionalized dextran coated on dextran silica beads led to the purification, with a glycine-HCl buffer, of another subclass of antibodies that recognized substituted dextran derivatives. Furthermore, these antibodies could be useful tools for in vitro and in vivo investigations using dextran derivatives as bio-active polysaccharides.     

Insoluble Glucan Formation by Leuconostoc mesenteroides B-1355

Leuconostoc mesenteroides B-1355 produced at least three glucosyltransferases (GTFs). We previously identified GTF-2 as alternansucrase and GTF-3 as fraction L dextransucrase. We here show that GTF-1 is a previously unreported sucrase that synthesized water-insoluble dextran. Our evidence consisted of the following. (i) GTF-1 was a major component and GTF-2 was a minor component of culture supernatant fractions, but supernatant fractions actively synthesized water-insoluble glucan. (ii) GTF-1 and culture supernatants produced an unusual high-pressure liquid chromatography pattern of malto-oligosaccharides that was not reproduced by GTF-2-GTF-3 mixtures. (iii) GTF-2, GTF-3, and GTF-2-GTF-3 mixtures did not synthesize insoluble glucan from sucrose. Nearly all of the alternansucrase in young (less than 17-h) cultures was associated with the cells.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

The autoactivation of factor XII (Hageman factor) induced by low-Mr heparin and dextran sulphate. The effect of the Mr of the activating polyanion.

Human Factor XII is known to undergo autoactivation in the presence of dextran sulphate of Mr 500,000. We have now studied the dependence of this reaction on the Mr of the dextran sulphate by using fractions resolved by gel filtration. We have found that autoactivation can be induced by dextran sulphate fractions with Mr as low as 3000, and there is a marked dependence of the rate constant of autoactivation on the Mr value. Fractions with Mr below 8000 gave very low rates of autoactivation; there was a sharp increase in the rate obtained when the Mr of the dextran sulphate was greater than 10,000. Various preparations of heparin were also able to support the autoactivation of Factor XII and gave a very similar relationship between molecular size and reaction rate. The data provide support for the hypothesis that the mechanism by which the 'surface' acts in contact activation involves the presence, on the same particle, of multiple binding sites for the proteins.     

Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran.

To determine the permselectivity characteristics of the glomerular capillary wall, known molecular size fractions of [3H]dextran, prepared by gel chromatography, were infused into normally hydrated Wistar rats, thus permitting simultaneous measurement of Bowman's space/plasma water (BS/P) and urine/plasma water (U/P) concentration ratios, along with glomerular pressures and flows. Since (BS/P)inulin = 1.01 +/- 0.01 SE(n = 34, radius = approximately 14 A) and since (BS/P)dextran/(BS/P)inulin equaled (U/P)dextran/(U/P)inulin for dextrans ranging in molecular radius from 21 to 35 A, these findings validate that dextrans are neither secreted nor reabsorbed. For dextran radii of 20, 24, 28, 32, 36, 40, and 44 A, (U/P)dextran/(U/P)inulin averaged 0.99, 0.92, 0.69, 0.42, 0.19, 0.06, and 0.01, respectively. In accord with theoretical predictions that these fractional dextran clearances should vary appreciably with changes in glomerular transcapillary pressures and flows, an increase in glomerular plasma flow rate, induced in these same rats by plasma volume expansion, resulted in a highly significant lowering of fractional clearance of all but the smallest and largest dextrans studied. These findings emphasize that fractional solute clearances alone are inadequate to describe the permselective properties of the glomerular capillary wall unless glomerular pressures and flows are also known. This sensitivity of fractional dextran clearance to changes in plasma flow indicates that dextrans are transported across the capillary not only by bulk flow but also to an important extent by diffusion.     

Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale

A detailed study of the influence of crude dextran on enzyme extractions in aqueous phase systems is presented in this article. The physical parameters of crude dextran, a purified T-500 fraction from Pharmacia, and a hydrolyzed crude dextran are compared and their influence on the phase system parameters investigated. Initially there is a drastic increase in the viscosity of the lower dextran-rich phase and a significant shift in the macroscopic structure of these phases, observed as the "gel-forming" properties of the dextran phases. The latter can be important for the partition of any enzyme by influencing the effect of phosphate concentration on the partition of proteins, although these experiments show that the partition coefficient of several enzymes is not much altered. The partition parameters allow the substitution of Dextran T-500 fractions by crude dextran or unfractionated, slightly hydrolyzed fractions. Using crude dextrans the performance and technical realization of enzyme extraction processes are demonstrated for pullulanase from Klebsiella pneumoniae and formate dehydrogenase from Candida boidinii.Both enzymes were recovered in comparable high yields. The equipment performance was quite good, as indicated by the high throughput values of the separators employed. Especially when using nozzle separators for phase separation there is a better performance in comparison to the Dextran T-500 fraction. No serious technical problems were encountered when replacing the expensive fractionated dextran with a crude dextran. In this way aqueous two-phase systems containing dextran become more feasible for enzyme purification from an economic point of view. The price of about 1.30 German marks (DM) per liter for a useful phase system already appears acceptable for the production of valuable intracellular enzymes.     

Immunochemical studies on dextrans: distribltion of alpha1 leads to 2 and alpha1 leads to 6 specific determinants of dextran NRRL B1397.

A rabbit anti-dextran serum was separated into two fractions, specific for α1→2 and α1→6 glucose linkages, respectively. Dextran NRRL B1397 containing α1→2 and α1→6 specific determinants was fractionated by step-wise precipitation with the α1→2 specific antibody fraction to see whether the α1→2 and α1→2 specific determinants were both in the same molecule. Most of the dextran fractions were shown to have two specificities by quantitative inhibition experiments with kojibiose and isomaltose as inhibitors. The precipitin lines in agar of dextran fractions with antibody fractions specific to α1→2 and α1→6 fused. These results strongly suggest that these two antigenic determinants are present in the same molecule.     

An improved procedure for the simultaneous purification in high yield of peroxisomes,lysosomes, and mitochondria from rat liver

Summary Peroxisomes, lysosomes, and mitochondria have been purified from rat liver by sucrose density gradient centrifugation without prior treatment of the animals with Triton WR-1339 or other detergents which cause hyperlipidemia. A crude organelle fraction was first prepared by differential centrifugation of a rat liver homogenate, this fraction contained approximately 70% of the mitochondrial, 40% of the peroxisomal, and 30% of the lysosomal marker enzymes measured in the homogenate. The crude organelle fraction was applied to the top of a sucrose density gradient and centrifuged. A clear separation of the organelles was obtained only when dextran was present in the gradients. Success or failure of the method was found to depend on the particular preparation of dextran used in the gradients. A method for subfractionating dextran was developed which yields dextran fractions that make the separations completely reproducible. Starting with a crude organelle fraction derived from 12 g of liver, approximately 85% of the mitochondrial, 70% of the peroxisomal, and 50% of the lysosomal activities were obtained as pure fractions. The organelle separation takes less than five hours to complete, it represents a substantial improvement over previous methods.     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary The activity and localization of -glucan phosphorylase in experimental canine glycogen-depleted heart tissue has been investigated with biochemical and histochemical methods using dextran as enzyme acceptor. Only linear, essentially unbranched, dextrans exhibit acceptor properties; highly branched dextrans are not suitable acceptors for the enzyme. Results of Michaelis-Menten constant measurements for the linear essentially unbranched dextran fractions used, indicate that the affinity of the enzyme for the non-reducing end group of the dextran molecule increases with increasing molecular weight of the acceptor.In the glycogen-depleted tissue of anoxic and ischaemic cardiac musculature there is a gradual inactivation of the enzyme during the ischaemic period. Shortly before total inactivation the affinity of the enzyme, especially for the lower molecular dextran fractions, is greatly reduced. Therefore, for the histochemical demonstration of phosphorylase activity in infarcted areas of the heart it is essential to use as acceptor an unbranched dextran fraction with a high average molecular weight.This investigation was partially supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).