Peter D.G. Dean

Protein purification using immobilised triazine dyes

This review attempts to identify proteins which selectively interact with immobilised triazine dyes such as Cibacron blue F3GA and Procion red HE 3B. Different support matrices are compared by examining the capacities of these dyes for proteins. Various approaches to the immobilisation of triazine dyes are considered together with the use of spacers. Some theories of the mechanism of protein retardation by immobilised dyes are discussed. A number of methods are suggested for the measurement of dye concentrations and for the modification of the binding of proteins to dye columns. The variety of elution methods is compared with a view to optimizing purifications. The scope of applications is reviewed as well as the choice of dye. Some advantages of triazine dyes over other affinity ligands are given. It is concluded that although no satisfactory mechanism for the binding of triazine dyes to proteins has yet been proposed, these dyes possess considerable potential for protein purification, particularly when applied on the large scale.

Use of Triazine dyes in the affinity chromatographic purification of alkaline phosphatase from calf intestine

Dye-ligand chromatography was examined as a method for the purification of alkaline phosphatase (EC.3.1.3.1). Forty six dye-Matrex Gels were assessed for their ability to bind alkaline phosphatase. Most dye adsorbents bound significant quantities of the enzyme. Three dye columns were examined in more detail for their selectivity using gradients of potassium chloride to desorb enzyme protein. Purification of alkaline phosphatase using Cibacron blue 3GA-Sepharose 6B chromatography was enhanced by using affinity elution. The best purifications (290-fold) were obtained using pulsed elution with the substrate alpha-naphthyl phosphate although the inhibitor, inorganic phosphate, was also useful (128-fold purification).

Some applications of insolubilised cofactors to the purification of pyridine nucleotide-dependent dehydrogenases

Abstract A study of the binding of several commercially available dehydrogenases to insolubilised NAD, NADP and AMP has revealed some interesting differences. The relative binding data provided can be used as a guide to the separation or purification of dehydrogenases from complex mixtures. The application of immobilised cofactors to the study of active sites is suggested.

The use of immobilized cibacron blue in plasma fractionation

Human serum albumin is a major contaminant in the isolation of many plasma proteins. It constitutes about 55% of the total plasma protein and is responsible for non-specific binding to many affinity columns. The use of Blue Dextran to abstract albumin from plasma was first reported by Travis and Pannell in 1974 [I] . The efficiency of the system was improved [2] by the use of the chromophore of Blue Dextran, viz. Cibacron Blue, fig.1, attached directly to agarose. We have already reported the influence of the matrix on the interaction of immobilized Cibacron Blue with albumin [3] . In this paper we describe the effect of pH on the adsorption of plasma by Cibacron Blue-Sepharose.

ELECTROPHORESIS: A NEW PREPARATIVE DESORPTION TECHNIQUE IN AFFINITY CHROMATOGRAPHY (AND IMMUNOADSORPTION)

A wide variety of methods exists for the elution of adsorbed material from affinity matrices which depend in many instances upon the nature of the interactions involved. For example, human serum albumin (HSA) can be eluted from Cibacron blue F3G-A-Sepharose using sodium thiocyanate, but the resulting HSA may not be clinically acceptable [ 11. Likewise, ferritin can be removed from immobilised ferritin antibody columns by elution with 6 M guanidine-HCl (pH 3.1), but the eluted protein is unstable and precipitates on standing [2]. In spite of these disadvantages, chaotropic reagents are frequently used to overcome such high affinity interactions. The adsorption of antibody to immobilised steroid is such that conventional elution methods have proved less than satisfactory. Buffers of pH 3.0, containing 10% dioxane were used [3] to elute testosterone antibodies from an affinity column, and oestradiol-6-(O-carboxymethyl)oxime [4] to displace oestradiol antibody from complexes with immobilised steroid. In both cases the recovery of high affinity antibodies was poor. The combination of electrophoresis and bio-specific interactions has been exploited analytically. The kinetics of the interaction of glycogen with various rabbit tissue phosphorylases [5], and of glycogen with potato phosphorylase and starch in human salivary amylase [6], have been studied by this method. Affinity electrophoresis has been used to investigate the interactions occurring in simple biospecific adsorption systems [7-l l] and the conditions for such analytical applications have been optimised [ 121.

Preparative electrophoretic desorption in the purification of human serum ferritin by immuno-adsorption

1. Introduction Since the original purification of ferritin by Laufberger [l] using cadmium salt precipitation a number of purification techniques have been described using various combinations of heat denaturation and differential precipitation with ammonium sulphate together with ultra-centrifugation and various forms of column chromatography [23] . The disadvantages of these methods are two-fold. Firstly, they usually involve several stages during which protein denatura- tion may occur and secondly, the yield is often very low, the latter being a major problem when working with a source of ferritin of low concentration, e.g., normal serum. Furthermore, ultra-centrifugation tends to select only those ferritin molecules with a relatively high iron content. This paper describes a method of ferritin purifica- tion which can be applied to both tissue and serum ferritin and combines the advantages of a one-step purification with high yield of recovered ferritin. The method involves an adsorption phase using a specific immuno-adsorbent combined with a previously described preparative application of electrophoretic desorption [4]. This desorption method was compared with more conventional methods. 2. Methods and materials Anti-human ferritin antibodies were raised in New Zealand white rabbits against human spleen ferritin which had been purified and assessed by established methods [5 ] . A horse

Affinity chromatography on columns containing nucleic acids

Chromatography on affinity adsorbents containing polynucleotide ligands dates back to 1968 when successful purification of micrococcal DNA poly- merase on a column containing DNA immobilized onto cellulose after irradiation with ultraviolet light was described by Rose Litman [1]. In the same year both Bruce Alberts [2] and Peter Gilham [3] independently suggested new methods for the prep- aration of adsorbents containing nucleic acids follow- ing earlier attempts mainly with oligonucleotides [4,5] or heavily damaged DNA [6,7] linked to different insoluble supports. Since that time, affinity chro- matography using immobilized nucleic acids has established itself as a useful technique [8-10] which has recently received diverse apphcations in molec- ular biology [11-20]. The following discussion summarizes the most significant methods of immobilization of poly- nucleotides and analyses applications of the affinity adsorbents obtained.

AFFINITY CHROMATOGRAPHY OF ENZYMES

The affinity chromatography of enzymes is reviewed in terms of the operational capacity of the ligand. Many groupspecific ligands function in a rather inefficient manner. Reasons for this inefficiency are examined and the apparent KAs calculated from frontal analysis data. The effect of changing the support matrix is presented as well as the variation of the latter with ligand concentration. The direct relation between the capacity and apparent KA is also shown. On the other hand, the capacities of two triazine-linked dyes, Procion Red and Cibacron Blue, are mugh higher than those of nucleotides. Cibacron Blue columns seem more effective in binding NAD +-dependent dehydrogenases whilst Procion Red columns are better suited to the purification of NADP +-dependent dehydrogenases. Yeast extract enzymes are separable using both blue and red columns with quite different elution profiles. The red column has been used to purify glutamate dehydrogenase from N. crassa. A novel method for the purification of L. casei dihydrofolate reductase on NADPH-Sepharose is described. A new method for the preparative electrophoretic desorption of proteins from affinity matrices and immunoadsorbents is illustrated.

The potential of Ultrogel®, an agarose-polyacrylamide copolymer, as a matrix for affinity chromatography

Received 13 March 1976 1. Introduction The selection of the inert support in the synthesis of adsorbents for affinity chromatography is constrain, ed by several factors. It should be stable to chemical and biological degradations, hydrophilic, easily substituted and uniform to enhance the chromato- graphic process. The comparative advantages of polyacrylamide, glass, cellulose, cross-linked dextrans and beaded agarose have been reviewed [1-3] ; currently the most popular support materials are polysaccharides, especially beaded agarose. Recently the microscopic structure of these polysaccharide matrices has been investigated both with respect to their ability to withstand various activation procedures, in particular cyanogen bromide [4], and also the distribution of the immobilised ligand throughout the matrix following coupling [5,6], which in itself may be a function of the CNBr activation [7]. In a previous communication [4] we reported that beaded cross-linked dextrans were susceptible to structural damage during CNBr activation, whilst beaded agarose was not obviously affected provided that vigorous stirring was avoided. However, microscopic inclusions and vacuoles were observed in approx. 5% of the commercially available agarose beads. Subsequently Gribnau et al. [8] reported the presence of similar particles in commer- cial preparations of agarose. The uncertain effect of

Improved antisera for the specific radioimmunoassay of oestetrol

5hydroxyoestriol) offers a possible solution to this problem, as its production is almost entirely dependent upon a functioning foetal liver [2] and initial investigations have indeed indicated that the measurement of this steroid may provide a useful index of foetal viability [3,4]. The antisera used in these earlier investigations were raised against an antigen in which the steroid was linked through the three-position and showed reasonable specificity for the D ring; the cross-reaction for oestriol being 2.5%