INTERFERENCE DUE TO NON-SPECIFIC ADSORPTION IN ION EXCHANGE CHROMATOGRAPHY OF PROTEINS: THE ROLE OF INITIAL SALT CONCENTRATION IN THE SEPARATION AND ANALYSIS OF LYSOZYME

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

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Studies on the protein-bound chondroitin sulphate of bovine cortical bone

Biochemical Journal ◽

10.1042/bj1070041 ◽

1968 ◽

Vol 107 (1) ◽

pp. 41-49 ◽

Cited By ~ 66

Author(s):

G. M. Herring

Keyword(s):

Ion Exchange ◽

Cortical Bone ◽

Chondroitin Sulphate ◽

Bone Sialoprotein ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Mild Conditions ◽

The Third ◽

Apparent Homogeneity

A fraction containing chondroitin sulphate, isolated from bovine cortical bone under mild conditions, was separated by ion-exchange chromatography into three fractions with apparent homogeneity on electrophoresis and ultracentrifugation. Two of these appeared to consist of chondroitin sulphate bound to a glycoprotein ‘core’ that had similarities to the bone sialoprotein described previously. The differences in composition of the two fractions were considered to be due to variation in the number or lengths of the polysaccharide chains. The presence of xylose and the alkali-lability of the bond between protein and polysaccharide suggested the presence of a xylosylserine linkage. The third fraction had the properties of a relatively pure chondroitin sulphate which contained a small amount of peptide. These fractions differed considerably from the protein–polysaccharide complexes of epiphysial and other cartilages, and their relevance to the possible role of glycosaminoglycans is discussed.

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Role of Methionine in the Initiation of the Biosynthesis of Bovine Proinsulin

Canadian Journal of Biochemistry ◽

10.1139/o73-097 ◽

1973 ◽

Vol 51 (6) ◽

pp. 783-788 ◽

Cited By ~ 1

Author(s):

C. C. Yip ◽

C. C. Liew

Keyword(s):

Amino Acid ◽

Ion Exchange ◽

Defined Medium ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Edman Degradation ◽

Tissue Slices ◽

Fetal Bovine ◽

Bovine Pancreas

Slices of fetal bovine pancreas were used to study the initiation of proinsulin biosynthesis. The pancreatic slices were incubated with radioactive methionine, phenylalanine, or leucine, in a defined medium. The incorporation of amino acid into peptides in the tissue slices was measured for 2–3 h. Two types of radioactive peptides, "free" and "blocked," were identified by ion-exchange chromatography. Most of the radioactive "blocked" peptides labelled with [3H]phenylalanine and [35S]methionine were hydrolyzed by proteases, except for about 20% of those labelled with [35S]methionine, which also showed higher resistance to acid hydrolysis.Microsomes were isolated from the tissue slices after incubation and were extracted with acid alcohol. The radioactive proteins in the extract were reacted with a solid immunosorbant against insulin. Analysis of the immunoadsorbed radioactive peptides by Edman degradation showed the presence of both methionine and phenylalanine as the N-termini. It was concluded that methionine was an initiating amino acid in the biosynthesis of bovine proinsulin.

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A study of the role of metallothionein in the inherited copper toxicosis of dogs

Biochemical Journal ◽

10.1042/bj2360409 ◽

1986 ◽

Vol 236 (2) ◽

pp. 409-415 ◽

Cited By ~ 17

Author(s):

D M Hunt ◽

S A Wake ◽

J F B Mercer ◽

D M Danks

Keyword(s):

Ion Exchange ◽

Liver Tissue ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Copper Accumulation ◽

Total Liver ◽

Liver Copper ◽

Rna Blots ◽

Dog Liver

The role of metallothionein (MT) was assessed in the copper-loading disease prevalent in Bedlington terriers. Fractionation of tissue supernatants over Sephadex G-75 showed that most of the additional cytosolic copper present in liver tissue of these dogs was bound to MT, and that substantially more MT-bound copper could be solubilized by detergent plus mercaptoethanol. Zinc contents were only slightly raised, although most of the extra zinc was associated with a 4000-Mr ligand. Ion-exchange chromatography revealed two isoproteins, MT1 and MT2, in all the dog liver samples examined. In Bedlington terrier liver, copper associated with both isoproteins was increased, although the increase for MT2 was greater than for MT1. The content of MT protein was also raised, although cell-free translations and RNA blots of total liver RNA showed that this increase was not associated with a rise in MT mRNA. The significance of these results to the mechanism of copper accumulation in the Bedlington terrier disorder is discussed.

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Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132008000400009 ◽

2008 ◽

Vol 51 (4) ◽

pp. 511-521 ◽

Cited By ~ 6

Author(s):

Alexandre Martins Costa Santos ◽

Jamil Silvano de Oliveira ◽

Eustáquio Resende Bittar ◽

Anderson Lourenço da Silva ◽

Marcos Luiz dos Mares Guia ◽

...

Keyword(s):

Ion Exchange ◽

Flow Rate ◽

Salt Concentration ◽

Mobile Phase ◽

Chemical Properties ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Purification Process ◽

Research Areas ◽

Physical Chemical

The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.

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