HPLC Anion-exchange Chromatography Research Articles

GLYCOSYLATION FEATURES OF FOLLITROPIN DELTA (REKOVELLE®) MORE RESEMBLE THOSE OF NATURAL HUMAN FSH COMPARED TO FOLLITROPIN ALFA (GONAL F®).

Compare the glycosylation properties of follitropin delta, from a human cell line, with those of natural human derived FSH and with follitropin alfa from a hamster cell line. Follitropin delta (Rekovelle®) produced in PER.C6® human derived cell line, follitropin alfa (Gonal f®) produced in CHO derived cell line and natural human urinary derived FSH (Bravelle®) were used in the study. Isoelectric Focusing (IEF), was used to resolve and profile the isoforms present in FSH preparations. IEF was carried out using a gel with a pH range between pH 3-7. Glycans were released from FSH preparations by enzymatic digestion with PNGase and derivatized with 2-aminobenzamide (2AB). Glycans were sequenced with specific exoglycosidases and analysed using normal phase HPLC and weak anion exchange chromatography. A more acidic isoform profile was obtained by IEF for follitropin delta and human derived FSH in comparison to follitropin alfa. Compared to follitropin alfa, the released glycans from follitropin delta and natural human FSH both show higher abundance of tri- and tetra-antennary forms and a lower abundance of the di-antennary forms. Sialylation of glycans on follitropin delta and natural human FSH comprises both α2,3 linked and α2,6 linked sialic acid, whereas follitropin alfa exhibits only the α2,3 linked form. The resemblance of follitropin delta glycans to the human derived FSH was further supported by the presence of N-acetylgalactosamine (GalNAc) and bisecting N-acetylglucosamine (GlcNAc) which are absent in CHO-derived FSH (Table). Follitropin delta glycosylation is richer and more complex compared to follitropin alfa with a higher proportion of tri- and tetra-antennary and charged glycans, as well as by the presence of α2,6-linked sialic acid, GalNAc residues and bisecting GlcNAc. In these ways follitropin delta shares numerous glycosylation features and more resembles natural human derived FSH.

Feasibility of asymmetric flow field-flow fractionation coupled to ICP-MS for the characterization of wear metal particles and metalloproteins in biofluids from hip replacement patients.

Hip replacements are used to improve the quality of life of people with orthopaedic conditions, but the use of metal-on-metal (MoM) arthroplasty has led to poor outcomes for some patients. These problems are related to the generation of micro- to nanosized metal wear particles containing Cr, Co or other elements, but the current analytical methods used to investigate the processes involved do not provide sufficient information to understand the size or composition of the wear particles generated in vivo. In this qualitative feasibility study, asymmetric flow field-flow fractionation (AF(4)) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was used to investigate metal protein binding and the size and composition of wear metal particles present in serum and hip aspirates from MoM hip replacement patients. A well-established HPLC anion exchange chromatography (AEC) separation system coupled to ICP-MS was used to confirm the metal-protein associations in the serum samples. Off-line single particle ICP-MS (spICP-MS) analysis was used to confirm the approximate size distribution indicated by AF(4) of the wear particles in hip aspirates. In the serum samples, AF(4) -ICP-MS suggested that Cr was associated with transferrin (Tf) and Co with albumin (Alb) and an unidentified species; AEC-ICP-MS confirmed these associations and also indicated an association of Cr with Alb. In the hip aspirate sample, AF(4)-ICP-MS suggested that Cr was associated with Alb and Tf and that Co was associated with Alb and two unidentified compounds; AEC analysis confirmed the Cr results and the association of Co with Alb and a second compound. Enzymatic digestion of the hip aspirate sample, followed by separation using AF(4) with detection by UV absorption (280nm), multi-angle light scattering and ICP-MS, suggested that the sizes of the Cr-, Co- and Mo-containing wear particles in a hip aspirate sample were in the range 40-150nm. Off-line spICP-MS was used to confirm these findings for the Co- and Cr-containing nanoparticles. Whilst limited in scope, the results are sufficient to show the interaction of ions with transport proteins and give an indication of particle size, providing useful pathological indices. As such, the methods indicate a new way forward for in vivo investigation of the processes which lead to tissue necrosis and hip loosening in patients with MoM hip replacements.

In vitro fermentation of prebiotic oligosaccharides by Bifidobacterium lactis HN019 and Lactobacillus spp.

The utilisation of various prebiotic oligosaccharides by probiotic strains of Bifidobacterium lactis, Lactobacillus rhamnosus and Lactobacillus acidophilus was investigated in order to determine the synbiotic potential of various prebiotic/probiotic combinations. Analysis by HPLC and high-performance anion-exchange chromatography of the cell-free medium taken during growth of the three probiotic bacteria showed differences in the consumption of the various oligosaccharides. Analysis of galactooligosaccharides showed that both L. rhamnosus and B. lactis consumed mostly mono- and di-saccharide, while L. acidophilus consumed oligosaccharides up to trisaccharide. Both B. lactis and L. acidophilus utilised fructooligosaccharides and inulin, but showed different patterns of oligosaccharide consumption. Only L. rhamnosus grew on β-glucan oligosaccharides and preferentially consumed the trisaccharide. The results indicate the synbiotic potential of the various probiotic/prebiotic combinations, particularly L. acidophilus/galactooligosaccharides, L. acidophilus/fructooligosaccharides or inulin and L. rhamnosus/β-glucan oligosaccharides.

Chemical composition and relative hydrophobicity of microbial exopolymeric substances (EPS) isolated by anion exchange chromatography and their actinide-binding affinities

Understanding the chemical and physical properties of microbially produced, actinide-carrying biopolymers is essential for the prediction of their relative binding affinities and particle-interaction propensities that are affecting the transport of actinide elements. In addition, they form the basis for their oceanographic applications, such as using POC/ 234Th ratios to determine organic carbon fluxes and for tracing upper ocean particle dynamics. Furthermore, environmental applications of bioflocculation and bioremediation can benefit from such improved understanding. Exopolymeric substances (EPS) in “attached” and “non-attached” forms, produced by two bacteria and one alga, were extracted and purified using improved procedures. These biopolymers produced by the two bacteria, which were previously shown to contain strong 234Th complexing ligands, were further fractionated by semi-preparative HPLC-anion exchange chromatography in order to relate Th(IV)-binding ability to chemical composition. Eluted fractions were further characterized at both semi-molecular and molecular levels. It was found that the EPS consisted of more than one biopolymer, with distinct monosaccharide and amino acid compositions, but very similar molecular weight (~ 25 kDa). 234Th binding strength was correlated to their uronic acid to organic carbon ratios, suggesting that uronic acids are the actual binding agents of 234Th, or more likely, proxy compounds for the actual macromolecular 234Th binding ligands. These compounds were, however, not pure uronic acids, but rather amphiphilic glycoproteins or proteoglycans. Hydrophobic contact areas (HCA) of “attached” EPS, as measures of their relative hydrophobicity and amphiphilicity, were higher than those of “non-attached” EPS, and all were highly correlated to the ratios of the FTIR areas of amide-I bands from proteins to those of C O C bands from carbohydrates. This suggests that the relative hydrophobicity of the carrier biopolymers, which can be responsible for gel formation, particle aggregation and bioflocculation, was mainly regulated by their relative protein to polysaccharide contents.

Analysis of galactoglucomannans from spruce wood by capillary electrophoresis

The aim of this study was to setup a method for detection and quantification of monosaccharide components in technical galactoglucomannas (T-GGM) from spruce wood using capillary zone electrophoresis (CZE). CZE technique was optimised regarding borate buffer concentrations, EOF modifier application, and system pH. Aqueous solution of T-GGM was chemically hydrolysed by sulphuric acid, in an autoclave. In this way obtained monosaccharides were derivatized with 4-amino benzoic acid ethyl ester via reductive amination using sodium cyanoborohydride. The results of the optimisation procedure showed that the borate buffers at lowest concentrations (100 and 200 mM) with acetonitrile addition as EOF modifier gave the optimal measurement results, as it showed sufficient separation at relatively short migration times. The amounts of single monosaccharide components in the T-GGM samples obtained by the optimised CZE procedure were practically the same in comparison to the results of the well established HPLC-anion exchange chromatography. On the basis of this research, it was concluded that the capillary zone electrophoresis is an efficient analytical procedure for the characterisation of galactoglucomannans derived from softwoods.

Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

Comparison of HPLC and Small Column (CDTect) Methods for Disialotransferrin

Current methods for determination of carbohydrate-deficient transferrin (CDT) are based on separation of the CDT fraction by ion-exchange chromatography on minicolumns and quantification by immunoassay. Alternatively, the transferrin isoforms can be separated by HPLC anion-exchange chromatography and quantified by absorbance. This method has been reported to improve the validity of CDT as a marker of chronic alcohol abuse. HPLC on either MonoQ or ResourceQ anion-exchange columns was used to separate and quantify isoforms of transferrin with detection at 460 nm. The result was expressed as the percentage of the disialo form (pI 5.7) of total transferrin (DST). The commercial CDTect assay was used as a comparison method. Serum samples from nondrinkers (n = 57), moderate drinkers (n = 77), and heavy drinkers (n = 139) were analyzed. In ROC analysis for differentiation between moderate and heavy drinkers, the area under the curve (AUC) for the HPLC method was 0.87 (95% confidence interval, 0.81-0.93), whereas that for CDTect was 0.72 (95% confidence interval, 0.64-0.80). At 90% specificity, the sensitivity of DST was 63% (95% confidence interval, 53-73%) compared with 33% (22-44%) for CDT. The reference interval of the HPLC method was 0.68-1.7%. The HPLC anion-exchange method for quantification of CDT provides substantially better separation between moderate and heavy drinkers than the CDTect method.

Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells

We have developed a simple and highly sensitive HPLC method for determination of cellular levels of sugar nucleotides and related nucleotides in cultured cells. Separation of 9 sugar nucleotides (CMP-Neu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) was examined by reversed-phase HPLC and high-performance anion-exchange chromatography (HPAEC). Although the reversed-phase HPLC, using an ion-pairing reagent, gave a good separation of the 12 nucleotides, it did not separate sufficiently the sugar nucleotides for quantification. On the other hand, the HPAEC method gave an excellent and reproducible separation of all nucleotides and sugar nucleotides with high sensitivity and reproducibility. We applied the HPAEC method to determine the intracellular sugar nucleotide levels of cultured Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five, BTN-TN-5B1-4) insect cells, and compared them with those in Chinese hamster ovary (CHO-K1) cells. Sf9 and High Five cells showed concentrations of UDP-GlcNAc, UDP-Gal, UDP-Glc, GDP-Fuc, and GDP-Man equal to or higher than those in CHO cells. CMP-Neu5Ac was detected in CHO cells, but it was not detected in Sf9 and High Five cells. In conclusion, the newly developed HPAEC method could provide valuable information necessary for generating sialylated complex-type N-glycans in insect or other cells, either native or genetically manipulated.

Purification and biochemical properties of dipeptidyl peptidase I from porcine skeletal muscle.

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.

Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor.

The human epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein having 11 potential N-glycosylation sites in its extracellular domain. N-Glycosylation is needed for proper membrane insertion, EGF binding and receptor functioning. The human epidermoid carcinoma A431 cell line secretes a soluble 105 kDa glycoprotein (sEGFR) that represents the extracellular domain of the membrane-bound form, and its glycosylation pattern has been investigated. After liberation of the oligosaccharides from sEGFR with PNGase F, the glycans were fractionated along different routes, including Concanavalin A affinity chromatography, anion-exchange chromatography, HPLC and high-pH anion-exchange chromatography. The oligosaccharide fractions were characterized by 500- and 600-MHz 1H-NMR spectroscopy and mass spectrometry (FAB, ESI, and MALDI-TOF). The oligomannose-type glycans range from Man5GlcNAc2 to Man8GlcNAc2 and account for 17% of the total carbohydrate moiety. Furthermore, di-, tri'- and tetraantennary complex-type structures are present, both neutral and (alpha2-3)-sialylated (up to tetrasialo), comprising 24 and 59%, respectively, of the total carbohydrate moiety. In this study, 32 new complex-type glycans are characterized containing the Le(x), Le(Y), and sialyl-Le(x) determinants, the bloodgroup A and H antigens, as well as the ALe(Y) determinant. This first comprehensive glycosylation study on a human nonrecombinant receptor shows the immense heterogeneity of the glycosylation of sEGFR.

Biochemical Characterization of a Beta Cell Membrane Fraction Antigenic for Autoreactive T Cell Clones

The two NOD-derived T cell clones, BDC-2.5 and BDC-6.9, are CD4+, Vβ4+, islet-specific, and diabetogenic. These two T cell clones show different response patterns to whole islet cell antigen, but were found to respond to the same fraction isolated from beta granule membranes. The clones were used to follow the antigenic activity in the biochemical purification of a beta cell membrane detergent lysate subjected to HPLC anion exchange (IEX) and size exclusion chromatography (SEC). Antigenic activity could be retained after lysis in only one detergent (octy1-β-glucoside) among several tested. In order to detect solubilized antigen, beta membrane proteins were covalently linked to microlatex beads prior to being added to T cell proliferation assays, a technique that eliminated detergent toxicity and resulted in increased assay sensitivity. To purify the antigen, membrane proteins were absorbed onto an anion exchange column and after elution using a salt gradient, activity for the clones was found in a fraction containing 0.15–0.2 M NaCl. Subsequent analysis of this material by size exclusion chromatography provided an apparent molecular weight of the antigen to be between 50 and 80 kDa. Further attempts to purify the protein by SDS-PAGE resulted in loss of antigenic activity. It is possible that the elusive nature of this protein is a clue to its importance as an autoantigen.

Isoforms and half-life of FSH from sheep with different reproductive states.

The glycoprotein hormone FSH comes in many different isoforms. In humans and rats the charges of the FSH isoforms vary with reproductive state and these affect the half-life of FSH in plasma. In this study we examined the charge heterogeneity of FSH in pituitary extracts from sheep with different reproductive states. Also the half-life of clearance of pituitary FSH from the different reproductive states was determined in mice. Pituitaries were collected from: anoestrous, luteal phase, follicular phase, early-pregnant and late-pregnant ewes, ewe lambs, ram lambs, rams during the breeding and non-breeding seasons and wethers (5 per group). After extraction, FSH isoforms were fractionated by HPLC anion exchange chromatography. The volume at which half of the FSH had eluted from the ion exchange column was determined (HP(50)). It was found that FSH isoforms from ewes (HP(50)=96.7+/- 1.3 ml (s.e.m. )) eluted later (P<0.01) than those from rams (HP(50)=82.3+/-1.3 ml) indicating that FSH isoforms in the ewes were more acidic than those from rams. There was a seasonal difference in ewes, with ewes in anoestrus (HP(50)=101.6+/-2.6 ml) having more-acidic (P<0.01) FSH isoforms than the ewes during the oestrous cycle (HP(50)=95.3+/-0.7 ml). There was an effect of age, with the FSH isoforms from cycling ewes (HP(50)=95.3+/- 0.7 ml) being more acidic (P<0.01) than those from ewe lambs (HP(50)=88.3+/-1.9 ml). There was an effect of pregnancy, with late-pregnant ewes (HP(50)=107.3+/- 1.6 ml) having more-acidic FSH isoforms (P<0.05) than those from anoestrous ewes (HP(50)=101.6+/-2.6 ml) and there was an effect of castration with the breeding season rams (HP(50)=80.7+/-1.4 ml) having more-acidic (P<0.05) FSH isoforms than wethers (HP(50)=74.0+/-0.5 ml). The half-life of pituitary FSH from animals in the different reproductive states was found to be negatively correlated with HP(50) (r(2)=0.56, P<0.01). The FSH isoforms from wethers were the least acidic and had the longest half-lives. Collectively, these findings show that in sheep, age, sex and reproductive state are all factors which influence the forms of FSH that are extracted from the pituitary gland. Moreover, these results demonstrate that FSH from sheep with the most-acidic FSH isoforms have the shortest half-life in plasma.

Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level.

The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16-20 mM) in the culturedecreased the apparent degradation of the EPO.

Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels.

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

Isolation of Lipophosphoglycans fromLeishmania donovaniAmastigotes

Leishmania donovani donovaniamastigotes, isolated from spleens of infected hamsters or axenically cultured, and promastigotes were comparatively examined for the expression of lipophosphoglycans (LPG). Parasites were metabolically labeled with [32P]phosphate, [3H]galactose, or [3H]mannose. Radiolabeled material was extracted with water/ethanol/diethylether/pyridine/NH4OH and purified further by gel filtration and hydrophobic column chromatographies. These radiolabeled compounds were identified as phosphorylated lipid-containing glycoconjugates. Mild acid treatment resulted in degradation of the glycolipids into low-molecular-weight fragments. All glycolipids isolated were susceptible to nitrous acid and phosphatidylinositol-specific phospholipase C treatments, as has been reported for theL. donovanipromastigote LPG. Moreover, glycoconjugates purified from the threeLeishmaniastocks were not susceptible to trypsin treatment. Acid hydrolysis of promastigote LPG resulted in a predominant [PO4-6galactose(β1,4)mannoseα1] fragment. In contrast, the main radiolabeled anionic fragments isolated from splenic and axenic amastigotes differed from that of promastigotes, as evidenced by the elution profiles obtained by HPLC anion-exchange chromatography. These cumulative results indicate that lipophosphoglycan molecules, structurally distinct from the previously characterized LPG of the promastigote stage, are being expressed byL. donovanisplenic and axenic amastigotes.

Eleven newly characterized xyloglucan oligoglycosyl alditols: the specific effects of sidechain structure and location on 1H NMR chemical shifts

Eleven previously uncharacterized oligosaccharides, each containing from seventeen to twenty glycosyl residues, were isolated from the xyloglucan produced by suspension-cultured Acer pseudoplatanus cells and characterized by 1H NMR spectroscopy, fast-atom bombardment mass spectrometry, and matrix-assisted laser-desorption mass spectrometry. The complex mixture of xyloglucan oligosaccharides released by endo-(1 → 4)- β-glucanase ( Trichoderma reesei) treatment of cell walls was similar to that released by digestion of the soluble xyloglucan present in the culture medium. The oligosaccharides were converted to oligoglycosyl alditols by borohydride reduction and purified by a combination of gel-permeation (Bio-Gel P-2) chromatography, normal-phase HPLC, reversed-phase HPLC, and high-performance anion-exchange (HPAE) chromatography. Eleven new oligoglycosyl alditols (along with several others that had been previously characterized) were isolated and characterized, allowing additional correlations between xyloglucan structure and specific chemical shift effects in the 1H NMR spectra to be determined. The correlations between structural and spectral features deduced in this study will facilitate the structural determination of a wide range of xyloglucans and their subunit oligosaccharides.

Selection of chromatographic protein purification operations based on physicochemical properties.

The charge characteristics of four proteins (conalbumin, ovalbumin, apotransferrin, and soybean trypsin inhibitor) were determined over a range of pH using an electrophoretic technique (titration curve). HPLC anion-exchange chromatography for each of the proteins was performed to investigate whether electrophoretic and chromatographic results could be correlated. It was found that charge density, estimated from the electrophoretic titration curves (as net charge divided by the protein molecular weight) in most cases correlates extremely well the retention of the protein in terms of time needed for the protein to be eluted from the column. This correlation was far better than that of net charge or that of surface charge (net charge/surface area). The performance of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration using preparative columns on an FPLC were compared. The ability of each method to separate the components of the mixture of the four proteins was assessed by calculating the resolution of the chromatographic results obtained for each pair of proteins. Using the concept of the deviation factor (DF) between individual properties of pairs of proteins (charge density, molecular weight, hydrophobicity), the separation efficiency factor (eta s) could be calculated. Resolution results showed that anion exchange was the best technique for separating the proteins followed by hydrophobic interaction chromatography and gel filtration. Calculations for efficiency, eta s, confirmed the superiority of anion-exchange chromatography as a separation method over gel filtration. For hydrophobic interaction chromatography, it was not possible to find a suitable parameter to express DF and, consequently, eta s. An appropriate way to express the hydrophobicity of a protein, as a physicochemical property, that can be used in the expression of DF should be investigated.

Purification by HPLC anion-exchange chromatography and some properties of a kinin-releasing enzyme from the venom of Agkistrodon halys ussuriensis

Purification by HPLC anion-exchange chromatography and some properties of a kinin-releasing enzyme from the venom of Agkistrodon halys ussuriensis. Toxicon 32, 157–163, 1994.—A kinin-releasing enzyme was isolated from the venom of Agkistrodon halys ussuriensis using a very convenient two-step procedure consisting of HPLC anion-exchange chromatography. The relative molecular mass was estimated as 24,000 by SDS gel electrophoresis and an isoelectric point of pH 3.7 was established by gel isoelectric focusing. The kinin-releasing enzyme resembled a typical tissue kallikrein in the values of such physiochemical parameters, in its thermal stability, in its lack of activity towards fibrinogen and casein, and in its behaviour with potential inhibitors. Thus, the enzyme can be classified as a kallikrein-like enzyme.

Purification and characterization of two high molecular weight hemorrhagic toxins from Crotalus viridis viridis venom using monoclonal antibodies

Q. Li, T. R. Colberg and C. L. Ownby. Purification and characterization of two high molecular weight hemorrhagic toxins from Crotalus viridis viridis venom using monoclonal antibodies. Toxicon 31, 711–722, 1993.—Two high mol. wt hemorrhagic toxins were purified from Crotalus viridis viridis venom using HPLC anion exchange and monoclonal antibody affinity chromatography following initial separation by HPLC with a preparative DEAE column. The fraction from the initial column having the highest hemorrhagic activity was used to immunize BALB/c mice for production of monoclonal antibodies. One of the monoclonal antibodies specifically recognized a single protein band of the immunizing fraction in native polyacrylamide gel electrophoresis. This monoclonal antibody was purified and conjugated to activated membranes for affinity purification of the antigen from the hemorrhagic fraction. Proteins eluted from the affinity membranes showed three bands in SDS-PAGE with apparent mol. wts of 68,000, 62,000 and 30,000. The 68,000 and 62,000 mol. wt proteins were further purified using an analytical DEAE column, and purity was demonstrated by the presence of a single band in SDS-PAGE and a single precipitation are against antibodies to Crotalus viridis viridis venom in immunoelectrophoresis. The 68,000 (p I of 8.5) and 62,000 (p I of 4.1) proteins are highly hemorrhagic metalloproteinases which cleave N, N-dimethylcasein and fibrinogen. Both are glycoproteins with the same galactos e- β(1–4)- N-acetylglucosamine side-chains linked to asparagine residues. Treatment of both toxins with glycosidase F blocked their hemorrhagic activities. Oxidation of the sugar moieties by periodate inhibited the hemorrhagic activity of the 62,000 toxin, but not of the 68,000 mol. wt toxin.

Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts

Preimplantation rabbit embryos collected at the early morula stage were cultured to blastocysts in the presence of [3H]inositol. The blastocysts were lysed, and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin-layer chromatographic separation of the lipid portion indicated that [3H]inositol was incorporated into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. HPLC anion-exchange chromatography indicated that [3H]inositol was incorporated into inositol phosphates, including the two second messengers, inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate, and also inositol monophosphate and inositol 1,4-bisphosphate. These results provide evidence that rabbit blastocysts may have an active phosphatidylinositol second messenger system, which may be responsive to intrauterine factors or intraembryonic paracrine factors.