Mookambeswaran A. Vijayalakshmi

A potential set up based on histidine hollow fiber membranes for the extracorporeal removal of human antibodies

Abstract Poly ethylene vinyl alcohol (PEVA) based hollow fiber membrane grafted with l -histidine has been found to be particularly useful for the preparative scale separation of immunoglobulin G and potential clinical applications. In this paper, we present a set-up designed for the extracorporeal removal of immunoglobulin G. Biochemical and hydrodynamic conditions were optimized so as to perform in vitro removal of 50% of plasma immunoglobulin G.

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems : erythrocytes as a model

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.

Antibody purification methods.

Antibodies (Abs) from the sera of patients with autoimmune diseases are reported to have different catalytic functions. Their recovery by effi cient purification methods is, therefore, a crucial step. This article reviews different available methods for their recovery and emphasizes a new approach, namely adsorbents with immobilized histidine, which allows a good purification both in yield and purity of Abs, with the addi tional advantage of using gentle elution conditions. This, in turn, will ensure the recovery of intact (nondenatured) catalytically functional Abs, directly from the sera.Antibodies (Abs) from the sera of patients with autoimmune diseases are reported to have different catalytic functions. Their recovery by effi cient purification methods is, therefore, a crucial step. This article reviews different available methods for their recovery and emphasizes a new approach, namely adsorbents with immobilized histidine, which allows a good purification both in yield and purity of Abs, with the addi tional advantage of using gentle elution conditions. This, in turn, will ensure the recovery of intact (nondenatured) catalytically functional Abs, directly from the sera.

Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system.

Immobilized metal ion affinity gel electrophoresis (IMAGE) has been applied to study the change of the surface histidines topography of RNase A when chemically glycosylated on exposed carboxylic groups with glucosamine using carbodiimide as cross-linker, under mild conditions. Two populations of glycosylated RNase A, one with a single glucosamine and another with two glucosamine attached, were obtained. These chemically glycosylated RNase A conserved about 80% of native enzymatic activity and their pls were increased in comparison to native RNase A. The chemically glycosylated RNase A showed dramatic enhancement for thermal stability, while proteolytic resistance was similar to that of native RNase A. Chemically glycosylated RNase A showed a slightly increased affinity to IDA-Cu(II) as compared to the native enzyme, which indicates that a conformational change and/or a decrease in steric hindrance around accessible surface histidines (His 12 or His 119 and His 105) has occured. IMAGE is a useful method to analyse subtle conformational changes in proteins which result in subtle changes in histidine accessibilities.

Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers : Sepharose 4B versus flat sheet and hollow fibre membranes

Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120,000 in protein-free solutions and 40 to 33,000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.

Structure-function relationship in glycosylated α-chymotrypsin as probed by IMAC and IMACE

Abstract Chemical glycosylation of bovine α-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of α-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of α-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods.

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: I. Study of the mechanistic aspects of adsorption for the separation of human serum albumin from its non-enzymatic glycated isoforms (advanced glycosylated end products)

The characteristics of albumin adsorption on histidyl-aminohexyl-Sepharose 4B were investigated. In particular, the adsorption capacity of the gel was studied as a function of conductivity and pH of the running buffer. The adsorption was maximum at low salt concentration around neutral pH, involving electrostatic and hydrophobic interactions. Kinetic aspects were also investigated. Dissociation constant (KD) and maximum capacity (Qx) were, respectively, estimated to be 4.5 x 10(-5) M (medium affinity) and 93.3 mg (high capacity) of human serum albumin per ml of adsorbent. According to these preliminary results, separation of HSA and its non-enzymatically glycated isoforms (conventionally named advanced glycated end products: AGEs) was achieved. Chromatographic potential of this separation tool is discussed.

Cell surface interactions with metal chelates.

We have explored immobilized metal ion affinity adsorption as a means of discrimination between cells and to assess partially the types of interaction that might contribute to the adsorption of cells on the such adsorbents. Erythrocytes from different sources were adsorbed on immobilized iminodiacetic acid charged with Cu2+, Ni2+ or Zn2+. The affinity of the human erythrocytes for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The adsorption capacity of the rat erythrocytes decreased in the following order: Zn2+ greater than Ni2+ greater than Cu2+. Pre-saturation of the columns with imidazole lead to the recovery of over 90% of the cells applied on the columns. Enzymic removal of sialic acid residues from the surface of erythrocytes has no effect on the adsorption-elution profiles of these cells on affinity adsorbents. These findings suggest that histidine residues localized on the cell surface are involved in the cell binding to the adsorbent. This new separation principle could be expanded to other types of cell. It could be used as a diagnostic tool and for separation, as well as for probing cell surfaces.

Selective affinity of l-histidine immobilized onto poly(ethylene–vinyl alcohol) hollow-fiber membranes for various oligoglucuronans: Influence of the degree of polymerization and the degree of substitution by acetate

Abstract A new species of oligoglucuronan was previously extracted from a polysaccharide crude fraction produced by a Sinorhizobium meliloti mutant strain, by chromatography on l -histidine immobilized onto poly(ethylene–vinyl alcohol) (PEVA) hollow-fiber membranes. Until now, only one specific species of oligoglucuronan was selectively adsorbed on the membranes with immobilized histidine. In this work, the specific membranes, commonly used to purify proteins, were tested on different non-ionic and anionic carbohydrate components. We determined only anionic oligosaccharides are selectively retained on these membranes. The affinity between the l -His–PEVA membranes and oligoglucuronans varying in the degree of polymerization and the degree of substitution by acetyl residues was studied.