Size-exclusion liquid chromatography and capillary electrophoresis of pollen allergens

Allergens from the pollen of Phleum pratense, Dactylis glomerata. Arrhenatherum elatius, Secale cereale, Lolium perrene and Festuca sp. were analysed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). SEC was used for the determination of the molecular masses of main allergens. A CE method, using either 150 mmol/1 phosphoric acid (pH 1.8) or a micellar system consisting of 50 mmol/l sodium dodecyl sulphate-20 mmol/l borate (pH 9.35), was developed as a rapid and efficient alternative to SEC, especially for process control of allergenic preparations. The results obtained by the two methods confirmed similarities in the structures of the studied pollen allergens.     

Characterization of acid phosphatase and phosphorylcholine hydrolase in adult Haemonchus contortus

An acid phosphatase (AP) and a phosphorylcholine hydrolase (PCH) were detected in excretory-secretory (ESP) products from adult Haemonchus contortus. The AP had a pH optimum of 4.5 and was inhibited by tartaric acid and sodium fluoride, but not by o-phenanthroline. The AP hydrolyzed paranitrophenol (pnp)-phosphate and to a lesser extent pnp-phenyl-phosphonate but did not hydrolyze diester substrates. Purified AP consisted of heterodimers with relative molecular weight (Mr) of 41.9 and 48.7 kDa and had a native molecular weight of 98 kDa by size-exclusion chromatography (SEC). The PCH had a pH optimum of about 9.5 and was inhibited by EDTA and o-phenanthroline but not by the specific phospholipase inhibitor D609. The specific activity of PCH in the ESP was approximately 25-fold less than that of AP. PCH also hydrolyzed 5'-thymidine monophosphate-pnp at a rate about 40% lower than pnp-phosphorylcholine but did not hydrolyze 3'-thymidine monophosphate-pnp. Partial purification of PCH suggests an Mr of 50.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 102 kDa by SEC. Both AP and PHC were secreted in vitro in a time-dependent manner and had their highest concentrations in the intestine. The results indicate that H. contortus adults secrete significant amounts of AP that might be a digestive enzyme. PCH is also an intestinal enzyme and is secreted in lesser amounts than AP. The PCH is probably not a phospholipase C but has some characteristics of a type I phosphodiesterase.     

Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures

A new approach was applied with the aim at producing plant protein hydrolysates less heterogeneous and less contaminated with nonpeptide substances than are the presently available digests. A significant reduction of nonprotein contaminants was achieved by extraction of the plant material, soy flour or wheat flour, with acetone prior to isolation of the protein. Enzymes of nonanimal origin, papain or Pronase, were used for protein hydrolysis. The components of the hydrolysates were resolved by low-pressure liquid chromatography. Separation of peptide fractions and of remaining nonpeptide contaminants was achieved using small-pore size-exclusion chromatography matrices, Sephadex G-15 or Biogel P-2. Individual peptide fractions, both from soy protein and from wheat gluten, varied substantially in their growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium. The highest enhancement of viable cell density in batch cultures was 180% of control, and the highest enhancement of final immunoglobulin concentration was more than 230% of control. The existence of marked differences in activity of individual peptide fractions leads to a suggestion that the hydrolysates may provide peptides exerting specific positive effects on cultured animal cells.     

Fractionation and characterization of a Polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography

A kind of regenerated cellulose gel (RCG) particles were treated by toluene to obtain particles with smaller mean pore size, then was mixed with the cellulose gel with pore size of 370 and 525 nm. A preparative size-exclusion chromatography (SEC) column (700 x 20 mm) packed with three gel particles was used to fractionate water-soluble polysaccharide (WEP) extracted from the sclerotia of Pleurotus tuber-regium by aqueous solution. The exclusion limit and fractionation range of the stationary phase of the preparative SEC were molecular mass 8 x 10(5) and 5 x 10(3) to 8 x 10(5), respectively. The calibration curve of the preparative SEC was represented as: log M=13.96-0.53 Ve. The WEP sample (weight-average molecular mass M(w)=2.2 x 10(4), polydispersity=2.4) was divided into three fractions with M(w) ranging from 1.4 x 10(4) to 3.4 x 10(4) by the preparative SEC column, and the fractions were characterized by gas chromatography GC, SEC combined with laser light scattering (SEC-LLS) and viscometry. The unfractionated WEP exhibited triple peaks due to different molecular mass, but each fraction exhibited single peak with the polydispersity of 1.1-1.8 in the SEC patterns. The results indicated that the preparative SEC was efficient for fractionation of polysaccharides having low molecular weight and for determination of their molecular mass.     

Characterization of polysaccharide-protein complexes by size-exclusion chromatography combined with three detectors

Two water-soluble samples (TM1 and TM2) were extracted from Pleurotus tuber-regium using 0.9% aqueous NaCl at 20 and 80 degrees C to obtain relatively low molecular mass fractions. The chemical structure of TM1 was analyzed to be a branched heteropolysaccharide-protein complex, and the sugar moiety was mainly beta-(1-->6)-, beta-(1-->4)-, and beta-(1-->3)-linked glucan containing galactose and mannose. TM2 was a branched polysaccharide-protein complex, and the sugar moiety was mainly beta-(1-->6)-, beta-(1-->4)-, and beta-(1-->3)-linked glucan. Preparative size-exclusion chromatography (SEC) and analytical SEC combined with three detectors were used to detect the TM1 and TM2 samples, confirming that the proteins were bonded to the polysaccharides. Furthermore, analytical SEC combined with online laser light scattering, differential refractive index detector, and UV to determine the components, weight-average molecular mass (M(w)) and chain conformation of the samples. The relatively low exponent values (nu) of S(2)(z)(1/2)=k(nu)M(w)(nu) for the samples in 0.15M aqueous NaCl at 25 degrees C suggested that TM1 and TM2 existed in compact sphere conformation in the aqueous solution. This work provided valuable information on the structure and chain conformation characterization of the polysaccharide-protein complex having relatively low M(w).     

Size exclusion chromatographic analysis of polyphenol-serum albumin complexes

Formation of water-soluble polyphenol-protein complexes was investigated by size-exclusion chromatography (SEC). The combination of (-)-epigallocatechin gallate (EGCG) and bovine serum albumin (BSA), which did not form a precipitate after the solutions were mixed, showed an SEC peak due to complex formation 2-24 h after mixing. Peak size of the complex varied with time, suggesting slow change of the conformation of the protein accompanied by complexation. Formation of the complex was substantiated by ultrafiltration of the mixture; the complex did not pass through a membrane with a 100,000 nominal molecular weight limit (NMWL). The SEC profile varied with the combination of compounds. The peaks due to the complexes showed that the apparent value of the number average molecular weight (M(n)) of the EGCG-BSA complex was 2.8x10(5), while that of a pentagalloylglucose (PGG)-BSA complex was 9.5x10(5) under the conditions used. Dimeric hydrolyzable tannins, oenothein B and cornusiin A, also caused changes in the SEC profile of BSA, although the combinations did not show peaks attributable to formation of such large complexes observed for EGCG and PGG. Procyanidin B3 and (+)-catechin did not cause changes in the SEC profile of BSA. With cytochrome c, EGCG did not show any chromatographic changes.     

Soluble fibrin-fibrinogen complexes as intermediates in fibrin gel formation

Oligomer formation in fibrinogen solutions following addition of thrombin was studied by addition of thrombin inhibitor at various times subsequent to thrombin, followed by size-exclusion chromatography (SEC) on a high-performance SEC column capable of resolving species of molecular weights less than or equal to 10(6). Peaks corresponding to species with 1, 2, 3, and 4 or more times the molecular weight of fibrinogen were detected and quantified via nonlinear least-squares curve-fitting procedures. The evolution of each of these peaks with time is well accounted for by a kinetic model in which the predominant component of each oligomeric molecular weight species is a linear complex of fibrinogen and fibrin. The observed predominance of trimeric over dimeric oligomers even at short times suggests that the thrombin-catalyzed release of the two A fibrinopeptides from a single molecule of fibrinogen is highly cooperative.     

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was perforrmed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600-18,000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.     

A novel protocol to accelerate dynamic combinatorial chemistry via isolation of ligand–target adducts from dynamic combinatorial libraries: A case study identifying competitive inhibitors of lysozyme

A novel protocol based on size-exclusion chromatography (SEC) and MS was established to accelerate dynamic combinatorial chemistry (DCC) in this study. By isolating ligand–target adducts from the dynamic combinatorial library (DCL), ligands could be identified directly by MS after denaturation. Three new inhibitors for lysozyme were discovered by this SEC–MS protocol in a case study. K_m Data for these new inhibitors was also determined.     

A deadenylase assay by size-exclusion chromatography

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.     

Size-exclusion chromatography of heparin oligosaccharides at high and low pressure

Recent findings on specific and non-specific interactions of glycosaminoglycans (GAGs) accentuate their pivotal role in biology and the call for improved sequencing tools. The present study evaluates size-exclusion chromatography (SEC) of heparin oligosaccharides at high and low pressure, requiring amounts as low as 0.2 microgram, using conventional UV detection after depolymerization with heparin lyases. Because of their high charge at physiological pH, SEC elution volumes of heparin oligosaccharides depend on both molecular size and charge repulsion from the matrix. As a consequence, SEC elution volumes of GAGs are smaller than those of globular proteins of similar molecular weight, and this might be exploited. Accordingly, larger heparin oligosaccharides are best separated according to their size at high ionic strength of the mobile phase (>30 mM); in contrast, disaccharides are best separated according to their charge at low ionic strength, compatible with on-line coupling to mass spectrometry. Optimized SEC affords separation of characteristic heparin trisaccharides that contain uronic acid at the reducing end and suggest cellular storage of heparin as a free glycan.     

Optimization of Lipase Purification by Rotating Annular Size-exclusion Chromatography

Crude porcine lipase was purified by continuous rotating annular size-exclusion chromatography. Sephadex G-75 was used as the size-exclusion packing material. Initial studies by this group on a similar unit have been reported [Genest et al. (1998) "Continuous purification of porcine lipase by rotating annular size-exclusion chromatography", Appl. Biochem. Biotechnol. 73, 215-230]. This article presents the results of optimization studies carried out on a modified unit. These modifications resulted in a better performance of the column and a higher throughput. Purification fold values of around 11 were achieved in most runs. The activity recovered was around 99% and the productivity was around 3 mg lipase/mg gel h.     

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ.mol-1 and 12.34 kJ.mol-1, in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.     

Labeling of insulin with non-radioactive I and application to incorporation of radioactive I for use in receptor-binding experiments by high-performance liquid chromatography

Conditions for the labeling of insulin with radioactive iodine isotopes were investigated by means of incorporation of non-radioactive ¹²⁷I into the peptide. Either the chloramine-T (CT) or lactoperoxidase-hydrogen peroxide (LPO) technique was applied and reversed-phase high-performance liquid chromatography (RP-HPLC) was used for analysis of the reaction products. The LPO method provided the ¹²⁷I-labeled peptide within 15–30 min, whereas the CT alternative yielded the labeled substrate even within 15 s. However, the latter reaction can only be controlled in a reproducible manner with difficulty and undesirad side-reactions became increasingly prominent when t a few seconds. In another experiment, the LPO technique was applied for radiolabeling insulin with ¹²⁵I. The product was first purified by size-exclusion chromatography (SEC) and then subjected to RP-HPLC. SEC yielded two peaks. The smaller one, which eluted at a slightly higher K_d value (accounting for about 14% of total radioactivity) predominantly consisted of material eluting at the column's void volume under the conditions of RP-HPLC, whereas the main SEC fraction (accounting for about 86% of total radioactivity) yielded a single peak, as shown by HPLC. The radioactive material attributable to the main SEC fraction revealed the expected receptor-binding properties, as evidenced by displacement experiments with non-radioactive insulin, as well as the action of tetradecanoyl phorbol acetate on the binding characteristics and thus indicating formation of a labeled hormone retaining biological activity.     

Problems connected with band-broadening in size-exclusion chromatography with dual detection

The sources of band-broadening (axial dispersion) in size-exclusion chromatography (SEC) of polymers and its influence on data obtained using dual detection of concentration and molecular weight are reviewed. Special attention is paid to the combination of a multiangle light-scattering photometer and a differential refractometer as detectors. The local polydispersity is discussed in the relation to the band-broadening as well as to heterogeneity of the polymer with respect to molecular weight and hydrodynamic volume.     

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.     

Purification and properties of glutaminase from Aspergillus oryzae

Glutaminase activity was found in a water extract of a wheat bran koji (extracellular fraction) of Aspergillus oryzae strains Lee-1, H-16 and MA-27-IM isolated from a commercial koji ssed for soy sauce fermentation, as well as in thier mycelia (intracellular fraction). Both the intracellular and the extracellular glutaminases were purified from strain MA-27-IM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weight of the intracellular and the extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various γ-glutamyl compounds besides l-glutamine but did not exhibit asparaginase activity. Further investigation of these preparations inidicated that these glutaminases possessed almost the same properties, suggesting their similarity.     

Mathematical Modeling of Size Exclusion Chromatography

A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk‐fluid phase, interfacial film mass‐transfer between the stationary and mobile phases, and diffusion of solutes within the macro pores of the packing particles. Differential equations of the process model were solved by the finite difference method. Characteristics of the column and the packing particles (bed void volume fraction, particle porosity, accessible particle porosity) were obtained experimentally, as well as retention times of different molecules with known molecular weights. Experiments were performed with two different columns containing two different packing materials, Superdex 75 HR 10/30 and BioSep SEC S2000, respectively. The model has been validated by comparing theoretical and experimental retention times for the different columns.     

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ x mol(-1) and 12.34 kJ x mol(-1), in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.     

NMR, cloud-point measurements and enzymatic depolymerization: complementary tools to investigate substituent patterns in modified celluloses

The substituent patterns of some chemically modified celluloses were characterized as a function of their size distribution, using size-exclusion chromatography coupled to both nuclear magnetic resonance spectroscopy (NMR) and cloud-point measurements. Intact and enzymatically hydrolyzed methyl cellulose (MC) was fractionated according to size, and the level of substitution of the fractions was measured off-line using NMR. Clouding behavior was also measured as a function of size. Clear differences between hydrolyzed and nonhydrolyzed samples were observed using both techniques. For samples that had been selectively hydrolyzed using cellulose-degrading enzymes, NMR data showed a direct link between the degree of degradation and the level of substitution. Differences in the clouding behavior highlighted changes in substituent levels and substituent patterns across the size distribution. The techniques gave valuable and somewhat complementary information on the substituent distributions of the samples before and after enzymatic hydrolysis.