Lars Sundberg

Preparation of adsorbents for biospecific affinity chromatography : I. Attachment of group-containing ligands to insoluble polymers by means of bifunctional oxiranes

Abstract A new method for preparing adsorbents for biospecific affinity chromatography is described. Bisoxiranes (e.g. 1,4-butanediol diglycidyl ether) have been used for the introduction of reactive oxirane groups into agarose, and for simultaneous stabilization of the gel by cross-linking. Optimal conditions for the activation and subsequent coupling of proteins, peptides and aliphatic and aromatic amines have been evaluated. Fractionation of different forms of trypsin on soya bean trypsin inhibitor agarose is described in order to illustrate the use of oxirane-agarose in biospecific affinity chromatography.

Studies on blood group substances: II. Coupling of blood group substance a to hydroxyl-containing matrices, including aminoethyl cellulose and agarose

Abstract 1. 1. Fully reversible and highly stable immunosorbents have been prepared by coupling of blood group substance A to agarose beads and aminoethyl cellulose using activation of carbohydrate with CNBr. 2. 2. Purification of anti-A phytohemagglutinin from Vicia cracca and human anti-A serum antibody by means of A-substance-agarose is reported. 3. 3. The amino group-carbohydrate linkage formed after activation of carbohydrate with CNBr has been found to be stable between pH 2 and 12. 4. 4. Due to their ease of preparation and the mild conditions required for their preparation, similar adsorbents may be of general value in immunochemistry.

Purification of L-asparaginase from E. coli by specific adsorption and desorption.

The effect of L-asparaginase in treatment of certain types of leukemia is now well established. The history of Lasparaginase as a therapeutic agent has been reviewed by Broome [l] . Recently, immunosuppressive properties of the enzyme have been reported [2,3]. A 900-fold purification of Lasparaginase from guinea pig serum was obtained by Yellin and Wriston [4] after a series of steps involving sodium sulfate fractionation, gel filtration on Sephadex G-200, and chromatography on DEAEcellulose and hydroxylapatite. The two distinct asparaginases EC-l and EC-2 of E. co/i have been purified from cell-free extracts by Campbell et al. [5] who used similar procedures, and by Roberts et al. [6] by means of ethanol fractionation, chromatography on DEAE-cellulose and CMSephadex, and polyacrylamide electrophoresis. Whelan and Wriston [7] reported a 2000-fold purification of E. coli B asparaginase by heat denaturation, gel filtration, chromatography on DEAE-cellulose and hydroxylapatite, and polyacrylamide electrophoresis. Large-scale purification of EC-2 for clinical use at present requires a costly multi-step process giving a product which is only partially pure. We believe that techniques developed in our laboratory [S-l l] might lead to a faster and simpler purification method based on specific adsorption and desorption on a matrix to which an inhibitor had been covalently bound. Reversible inhibition by D-asparagine or Lasparaginase from Micobactetium phlei was reported by Grossowicz and Halpern [ 121, and rat liver and guinea pig liver asparaginases were found by DeGroot and Lichtenstein [ 131 to be inhibited by a series of ol-A’-alkyl derivatives of DL-asparagine. Mor and Lichtenstein [ 141 showed that carbobenzoxy derivatives of Land DL-phenylalanine, Ltyrosine, and L-

Affinity chromatography of carbonic anhydrase

During the last years several examples have been presented showing how the covalent attachment of an enzyme inhibitor, cofactor, or substrate, to an inert matrix can provide an adsorbent useful for the selective purification of an enzyme from a complex mixture [l-7] . The enzyme carbonic anhydrase is strongly and specifically inhibited by aromatic and heterocyclic sulfonamides [8,9]. These inhibitors would be expected to be useful in designing biospecific adsorbents for the enzyme. The present paper describes the chromatography of human and bacterial carbonic anhydrases on an adsorbent consisting of Sephadex or Sepharose to which sulfanilamide had been coupled. After adsorption of the enzyme to the column elution can be brought about in a predictable manner by agents that are known to act competitively with sulfonamides. The human erythrocyte enzyme, as described elsewhere [9] , consists of a mixture of two forms that differ from each other in the structure of the binding site for inhibitors. This difference can be utilized to separate the two isoenzymes by selective displacement after adsorption on a chromatography column. Preliminary results of this investigation have been communicated elsewhere [4] and later Whitney reported similar experiments [lo] .

Purification and properties of tyrosinase inhibitor from mushroom

The control of the enzymic oxidation of phenols has practical significance in the therapy of melanomas [l] and leprosy [2], in senescence [3] and food processing [4]. Extracts inhibitory to mushroom tyrosinase have been obtained from mushrooms [5,6], from guinea pig skin [7,8], from the fungus Ductylium dendriodes [9] and from potato tubers [lo] . The inhibitors in these extracts have all been described as proteins. Many of their properties however, are dissimilar indicating different entities even from the same sources [7]. Tyrosinase (EC 1.10.3.1) is widely distributed in plants and animals and its activity on phenolic substrates becomes particularly apparent in injured tissues of apple, banana and potato. This paper describes the properties of a tyrosinase inhibitor purified by column and affinity chromatography.

Purification of T4 phage by adsorption on polylysine agarose

Purification of complex viruses usually involves a series of fractionation procedures, necessarily performed under mild conditions to prevent destruction of functional structures of the virus. Density gradient centrifugation [l] and zonal ultracentrifugation [2], have been used for purification of the complex influenza virus, which has also been purified by electrophoresis in 0.16% agarose [3] . Another mild method is distribution in a two-phase system which has been used for purification of e.g. T2 phages [4] . Chromatography procedures have been employed as well: by selecting conditions for adsorption and elution of virus on calcium phosphate it is possible to remove contaminants [5]. Recently, the successful isolation of Aleutian Mink Disease virus by biospecific affinity chromatography, involving an immunoadsorption technique, has been reported [6] . Our aim has been to introduce another mild, simple, and more specific method for purification and concentration of T4 phages from the lysates of T4-infected E. coli. Our method involves adsorption and desorption of T4 phages from polylysine agarose. The principle is based on the observations by Katchalski that polylysine in solution interacts with viruses, bacteria, etc. [7] . The homogeneity and the biological activity of the samples obtained have been analyzed by plaque assays, gel filtration, and electron microscopy.

Chemical fixation of elastase to agarose

Porcine pancreatic elastase (Pancreatopeptidase, EC 3.4.4.7, 2 X crystallized in water) and elastin Congo Red were obtained from Seravac (England); Trypsin, Chymotrypsin, N-Acetyl-L-tyrosine ethylester (AcTyrOEt), Tosylargine ethylester (TAME) from Sigma (USA); N-Acetyl-L-alanyl-Lalanyl-L&nine methylester (AcAla30Me) from Sigma (USA); Agarose (Sepharose 2B, 4B, 6B) from Pharmacia (Sweden) and cyanogen bromide was purchased from Fluka (Switzerland).