Affinity Chromatography Elution Research Articles

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

CoVaccine HT™ adjuvant is superior to Freund's in eliciting ovine polyclonal antibodies against human tumor necrosis factor-alpha.

Passive immunotherapy using polyclonal antibodies plays an important role in preventing and treating antigenic and pathogenic diseases. Polyclonal antibodies are used for therapeutic, diagnostic and investigational purposes, with adjuvants employed to enhance the immune response against proteins that are poorly antigenic or self-antigens. This study aimed to optimize current immunization methods by evaluating the novel adjuvant CoVaccine HT™ against the established Freund's at producing ovine polyclonal antibodies against pro-inflammatory cytokine human recombinant tumor necrosis factor alpha (TNF-α). Castrated male Aberfield cross sheep were immunized with TNF-α in CoVaccine HT™ or Freund's adjuvant. The binding titer of antibodies for TNF-α and neutralization titer were determined in vitro, as well as the strength of antibody binding by a simple small scale affinity chromatography elution experiment. Animal welfare was monitored through inspection of immunization site reactions at regular time points and graded according to reaction size. The second part of the study looked at re-immunization using Freund's adjuvant alone every 4- or 8-weeks. Freund's generated significantly higher antibody binding titers than CoVaccine HT™ but were less effective at neutralizing TNF-alpha which is a better indicator of functional potency. CoVaccine HT™ also caused fewer immunization site reactions, while no statistical difference was observed in the binding strength of antibodies. Re-immunization every 4- and 8-weeks showed no statistical difference. This study provides evidence that CoVaccine HT™ is superior to Freund's adjuvant for the production of antibodies to TNF-α, and supports the use of this alternative adjuvant for clinical and experimental use. The outcomes gained through this study are applicable to passive and active immunotherapy for the generation of polyclonal antibodies in human and veterinary medicine.

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets.

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.

Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives

Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.

Overexpression of myosin-IIB in the brain of a rat model of streptozotocin-induced diabetes

The Ca2+/calmodulin complex interacts with and regulates various enzymes and target proteins known as calmodulin-binding proteins (CaMBPs). This group of proteins includes molecular motors such as myosins. In this study, we show that non-muscle myosin-IIB is overexpressed in the brains of diabetic rats. We isolated CaMBPs from the brains of non-diabetic rats and rats with streptozotocin-induced diabetes and purified them by immobilized-calmodulin affinity chromatography. The proteins were eluted with EGTA and urea, separated by SDS-PAGE, digested and submitted to peptide mass fingerprinting analysis. Thirteen intense bands were found in both types of brains, two were found exclusively in non-diabetic brains and four were found exclusively in diabetic brains. A large fraction of the eluted proteins contained putative IQ motifs or calmodulin-binding sites. The results of the myosin-IIB affinity chromatography elution, western blot and RT-PCR analyses suggest that myosin-IIB protein and mRNA are expressed at high levels in diabetic brains. This is the first study that has demonstrated differential expression of CaMBPs in diabetic and non-diabetic brain tissue through a comparative proteomic analysis, and it opens up a new approach to studying the relationship between the expression of myosins in the brain, hyperglycemia and intracellular calcium regulation.

DNA Transcription and tRNA Ligase: the Work of John Abelson

Saccharomyces cerevisiae tRNA Ligase. Purification of the Protein and Isolation of the Structural Gene(Phizicky, E. M., Schwartz, R. C., and Abelson, J. (1986) J. Biol. Chem. 261, 2978–2986)Yeast RNA Ligase Mutants Are Nonviable and Accumulate tRNA Splicing Intermediates(Phizicky, E. M., Consaul, S. A., Nehrke, K. W., and Abelson, J. (1992) J. Biol. Chem. 267, 4577–4582) Saccharomyces cerevisiae tRNA Ligase. Purification of the Protein and Isolation of the Structural Gene (Phizicky, E. M., Schwartz, R. C., and Abelson, J. (1986) J. Biol. Chem. 261, 2978–2986) Yeast RNA Ligase Mutants Are Nonviable and Accumulate tRNA Splicing Intermediates (Phizicky, E. M., Consaul, S. A., Nehrke, K. W., and Abelson, J. (1992) J. Biol. Chem. 267, 4577–4582) John Norman Abelson was born in Grand Coulee Dam, Washington, in 1938. He attended Washington State University and graduated in 1960 with a bachelor's degree in physics. He then moved east to pursue graduate studies at Johns Hopkins University, where he earned a doctorate in biophysics in 1965. After that, Abelson moved again, this time to Cambridge, England, to do a postdoctoral fellowship in the Structural Studies Division at the MRC Laboratory of Molecular Biology. There he worked with Sydney Brenner and Francis Crick studying DNA transcription. In 1968, Abelson returned to the United States to accept a position as assistant professor in the chemistry department at the University of California, San Diego. He was promoted to associate professor in 1973 and professor in 1977. In 1982, Abelson joined the faculty of the California Institute of Technology as a professor of biology. He chaired the Division of Biology and became the George Beadle Professor of Biology in 1991. At Caltech Abelson continued to study transcription, focusing on tRNA precursor splicing and intron excision. In the early 1980s he was able to partially purify tRNA ligase from Saccharomyces cerevisiae (1Greer C.L. Peebles C.L. Gegenheimer P. Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing..Cell. 1983; 32: 537-546Abstract Full Text PDF PubMed Scopus (178) Google Scholar). A few years later, he and his colleagues purified the protein to near homogeneity, which is the subject of the first Journal of Biological Chemistry (JBC) Classic reprinted here. Using affinity elution chromatography, they showed that tRNA ligase is a single polypeptide of 90 kDa that also contains polynucleotide kinase and 2′,3′-cyclic phosphate 3′-phosphodiesterase activities. They also determined the amino-terminal sequence of the purified protein and used this information to isolate the gene encoding tRNA ligase by hybridization of an appropriate set of mixed oligonucleotides to a library of yeast DNA. In the second JBC Classic reprinted here, Abelson and his colleagues continue their ligase studies, demonstrating that not only is tRNA ligase critical for tRNA splicing in the yeast cell, but it is also essential for cellular survival. They created a haploid yeast strain carrying a chromosomal deletion of the ligase gene and found that it was viable only if ligase protein was supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase was repressed, the cells accumulated endonuclease-cut, unligated half-molecules and eventually died. Two temperature-sensitive ligase mutant yeast strains also accumulated unligated half-molecules at nonpermissive temperatures. Abelson retired in 2002 and is currently George W. Beadle Professor of Biology, Emeritus at Caltech. He is also president and executive director of Agouron Institute, a non-profit research foundation, which he co-founded in 1978. Abelson also participated in the creation of Agouron Pharmaceuticals, Inc., a small San Diego firm that used rational drug design to develop Viracept, the leading drug used for controlling HIV infections. In recognition of his contributions to science, Abelson has received many awards and honors including the Washington State University (WSU) Distinguished Alumnus Award from the College of Sciences (1993), the WSU Alumni Achievement Award (1995), and the WSU Regents' Distinguished Alumnus Award (2004), as well as election to the National Academy of Sciences (1985), the American Academy of Arts and Sciences (1985), and the American Philosophical Society (2001). He has served as an editor of Methods in Enzymology for the past 15 years and was an assistant editor for Analytical Biochemistry (1980–1987) and a member of the JBC editorial board. He is also a past president of the RNA Society.

The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants.

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.

Regulation of AP-2-Synaptotagmin Interaction by Inositol High Polyphosphates

The inositol high-polyphosphate series (IHPS) inhibits neurotransmission through binding to the second C2 domain of synaptotagmins I and II(Syt), synaptic vesicle membrane proteins. We have revealed that several proteins, including α adaptins which are specific subunits of clathrin assembly protein, AP2, were eluted from mouse brain by affinity elution chromatography from the C2 domains of Syt II-immobilized Sepharose using 50 μM of InsP6. The interaction between Syt II and AP2 was more markedly inhibited by IHPS than by the same concentration of InsP3. Limited digestion of mouse crude synaptosomal fractions with trypsin revealed different cleavage patterns in the presence and absence of 50 μM InsP6. These results suggest that IHPS-binding to the C2B domain of synaptotagmin alters the state of protein–protein interaction including the synaptotagmin-AP2 interaction, possibly resulting in the inhibition of events involved in the synaptic vesicle trafficking.

Storage stability of the solution formulation of sCD4 determined by DSC in comparison with two functional assays

Differential scanning calorimetry (DSC) was used, in conjunction with two functional assays that monitor binding, in a storage stability study on a protein of pharmaceutical interest, the soluble form of the T-lymphocyte multidomain surface receptor, sCD4. DSC monitored structural changes in binding and non-binding domains. ELISA, using the monoclonal antibody OKT4a, and frontal elution affinity chromatography, using the HIV surface glycoprotein, gp120, monitored function of the binding domain. The stability of sCD4 in a solution formulation was followed for up to 30 days at five differentpHs ranging from 5.0 to 7.9 and five different temperatures ranging from −70‡C to 40‡C. While the overall trends observed with the three techniques were the same, the ELISA data were somewhat less reproducible than those for the other methods. Furthermore, the results suggest that DSC is more sensitive to structural changes that would reduce the protein's bioactivity. The results of this study indicate DSC's utility, in conjunction with quantitative functional analysis, in the formulation of protein-containing pharmaceuticals or foods, especially those containing multiple-domain proteins.

Inositol hexakisphosphate binds to clathrin assembly protein 3 (AP-3/AP180) and inhibits clathrin cage assembly in vitro.

We have isolated an inositol hexakisphosphate binding protein from rat brain by affinity elution chromatography from Mono S cation exchange resin using 0.1 mM inositol hexakisphosphate (InsP6). The amino acid sequences of six tryptic peptides from the protein were identical to the sequences predicted from the cDNA encoding a previously isolated protein designated as AP-3 or AP180. This protein is localized in nerve endings and promotes assembly of clathrin into coated vesicles. The isolated protein-bound InsP6 with a dissociation constant of 1.2 microM and a stoichiometry of 0.9 mol of InsP6 bound/mol of AP-3. Recombinant AP-3 expressed in Escherichia coli also bound InsP6 with a similar affinity. InsP6 inhibited clathrin cage assembly mediated by AP-3, in an in vitro assay, but had little effect AP-3 binding to preformed cages. We speculate that InsP6 and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation.

Purification of deoxyhypusine synthase from Neurospora crassa to homogeneity by substrate elution affinity chromatography.

Deoxyhypusine synthase is an NAD(+)-dependent enzyme that catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor by using spermidine as the substrate. Deoxyhypusine synthase bound tightly to 1,12-diaminododecane-agarose and could be eluted selectively by spermidine. This finding enabled us to develop a simple two-column procedure to purify deoxyhypusine synthase from Neurospora crassa to apparent homogeneity. The purified enzyme had a specific activity of 130,000 units/mg of protein, representing a 64,000-fold purification from cell extracts. Size exclusion chromatography indicated that the native enzyme had a molecular mass of 180 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme showed a single band at the 40-kDa position, suggesting that Neurospora deoxyhypusine synthase is a homotetramer. Deoxyhypusine synthase appeared to be hydrophobic and required non-ionic detergent such as Tween 20 to stabilize the activity. Treatment of the enzyme with sulfhydryl reagents resulted in a complete loss of activity. Inclusion of NAD+ reduced the inactivation rate by manyfold, indicating the presence of -SH groups at or near the active site. Partial amino acid sequences of four peptide fragments that cover about one quarter of the enzyme were obtained for cDNA and genomic cloning work.

Hydrolysis of phosphatidylinositol 3,4-bisphosphate by inositol polyphosphate 4-phosphatase isolated by affinity elution chromatography.

Inositol polyphosphate 4-phosphatase is a monomeric 110-kDa protein that hydrolyzes two substrates in the inositol phosphate pathway. Inositol 3,4-bisphosphate is converted to inositol 3-phosphate, and inositol 1,3,4-trisphosphate is converted to inositol 1,3-bisphosphate. We have exploited the fact that inositol hexasulfate inhibits the enzyme to devise an affinity elution scheme from a Mono S cation exchange column that resulted in an 11,300-fold purified preparation of rat brain 4-phosphatase. The resulting 4-phosphatase hydrolyzed phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3-phosphate with a first order rate constant 120-fold greater than that for inositol 3,4-bisphosphate and 900-fold greater than that for inositol 1,3,4-trisphosphate. This is now the third example wherein the same enzyme hydrolyzes both an inositol lipid and its analogous inositol phosphate.

2] Parameter estimates from nonlinear models

This chapter focuses on parameter estimates from nonlinear models. The methods require some extra computing after the model has been fitted to a data set but the computing is efficient and easily accomplished. Fitting nonlinear models to data relies heavily on procedures used to fit linear models. The chapter presents a review of fitting linear models, including how to assess the quality of parameter estimates for such fits and discusses fitting nonlinear models and application of linear model methods for assessing the quality of parameter estimates for nonlinear models. The chapter also discusses a more accurate and valid procedure for characterizing the behavior of estimates of parameters in nonlinear models, illustrating the approach and the insights it gives using a model for frontal elution affinity chromatography and a compartment model. Profile plots can be extremely useful in nonlinear model building because they remove the gross dangers involved when using linear approximation standard errors and confidence regions.

Molecular basis of the association of arterial proteoglycans with low density lipoproteins: Its effect on the structure of the lipoprotein particle

Modifications of low density lipoproteins (LDL) that enter the arterial intima appear to be responsible for their eventual extracellular and intracellular accumulation during atherogenesis. Some of these modifications seem to be the result of LDL association with intimal chondroitin sulphate-rich proteoglycans (CSPG). We have used frontal elution affinity chromatography, binding and competition experiments with synthetic segments of apoB-100 to better define the ligand regions for the LDL-CSPG complexes. The minimum structural requirement for recognition by the CSPG appears to be a hydrophilic nine-residue amino-acid segment with five lysine and arginine residues. Analysis of other similar regions in apoB-100 and other glycosaminoglycan-binding proteins suggest that besides a cluster of positively charged amino-acids, the presence of hydroxyl-containing residues favours the association with sulphated proteoglycans. With controlled proteolytic hydrolysis, we found that the interaction of LDL with CSPG modifies the surface accessibility of a apoB-100 segments containing arginine and lysine. Because these apoB-100 domains may also be involved in cell-receptor binding, the CSPG-induced modifications could be the structural explanation for the observed increase in cellular uptake of proteoglycan-modified LDL.

Improved resolution of glycoproteins by chromatography with concanavalin A immobilized on microparticulate silica via temperature-programmed elution.

The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyldiimidazole), as well as the effect of including a monosaccharide in the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.

Identification of Apo B-100 segments mediating the interaction of low density lipoproteins with arterial proteoglycans.

The interactions of low density lipoprotein (LDL) and apolipoprotein (apo) B-100 segments with chondroitin-6-SO4 rich aortic proteoglycans aggregate (CSPG) were studied by using quantitative frontal elution affinity chromatography. The affinity of the agarose-CSPG was higher for LDL than for very low density lipoprotein and high density lipoprotein was not bound. LDL from different individuals had dissociation coefficients (Kd) from 28 to 179 nM. Experiments with tryptic hydrolysates of apo B suggested that the capacity of LDL to bind with CSPG resides in the protein. Nine apo B-100 hydrophilic peptides, 12 to 26 amino acids long, were selected, and three were found to interact with the agarose-bound CSPG: apo B P-1 (LRKHKLIDVISMYRELLKDLSKEA, residues 4230 to 4254), apo B P-2 (RLTRKRGLKLATALSLSNK, residues 3359 to 3377), and apo B P-11 (RQVSHAKEKLTALTKK, residues 2106 to 2121). These peptides competed with LDL for binding to the agarose-bound and soluble CSPG; apo B P-2 was the most effective. This correlates with Kd values: 63, 86, and 82 microM for apo B P-2, P-1, and P-11, respectively. The peptides shared an excess of positive-charged side chains. Apo B P-2 belongs to the lys- and arg-rich, LDL-receptor domain. Apo E also binds to the agarose-proteoglycan. The results suggest that apo B regions with sequences and charge distributions analogous to those of residues 3359 to 3377, 4230 to 4254, and 2106 and 2121 are among those responsible for the interaction of LDL with intima-media CSPG.