STUDIES WITH STAPHYLOCOCCAL TOXINS: VII. SEPARATION OF A PROTEOLYTIC ENZYME FROM ALPHA HEMOLYSIN

1963 ◽  
Vol 9 (5) ◽  
pp. 697-702 ◽  
Author(s):  
J. Robinson ◽  
F. S. Thatcher
Keyword(s):  
Ion Exchange ◽  
Ethyl Alcohol ◽  
Zinc Acetate ◽  
Proteolytic Enzyme ◽  
Potato Starch ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sheep Erythrocytes ◽  
Alpha Hemolysin ◽  
Zone Electrophoresis

Certain preparations of alpha hemolysin induced dissolution of rabbit and sheep erythrocytes and hydrolyzed casein. Components which were independently hemolytic and proteolytic were separated by the following sequence of procedures: precipitation with zinc acetate and ethyl alcohol at approximately −5 °C and pH 4.0, ion exchange chromatography with carboxymethylcellulose, and zone electrophoresis with granules of potato starch. The comparative lytic activity of the hemolytic component against erythrocytes of the rabbit and of the sheep was 19:1. No separation could be effected between these two hemolytic properties.

STUDIES WITH STAPHYLOCOCCAL TOXINS: V. POSSIBLE IDENTIFICATION OF ALPHA HEMOLYSIN WITH A PROTEOLYTIC ENZYME

1960 ◽  
Vol 6 (2) ◽  
pp. 183-194 ◽  
Author(s):  
J. Robinson ◽  
F. S. Thatcher ◽  
Jeannine Montford
Keyword(s):  
Proteolytic Enzyme ◽  
Divalent Cations ◽  
Tetraacetic Acid ◽  
Sheep Erythrocytes ◽  
Alpha Hemolysin ◽  
Apparent Loss ◽  
Zone Electrophoresis ◽  
Chromatographic Procedure ◽  
Different Cultures ◽  
Hydrolyze Casein

Alpha hemolysin has been separated from many of the other components present in culture filtrates of four strains of Staphylococcus aureus using a column chromatographic procedure with carboxymethyl-cellulose as the selective adsorbent, and graded levels of phosphate buffer at pH 6.0 to provide selective elution. The preparation of alpha hemolysin obtained in this manner hemolyzed rabbit and sheep erythrocytes, induced a dermonecrotic reaction in rabbits, was lethal to mice, and was proteolytic. The hemolytic activity of the preparation was stimulated by ethylenediamine tetraacetic acid, presumably by removing toxic ions from solution. Divalent cations inhibited activity of the alpha hemolysin, but stimulated the activity of a distinctive sheep hemolysin, which is shown to be a separate entity.Alpha hemolysin, obtained by the chromatographic procedure, and subjected further to resolution by zone electrophoresis retained its capacity to induce dermonecrosis, to hemolyze rabbit erythrocytes, and to hydrolyze casein. Apparent loss of the capacity of the preparation to hemolyze sheep erythrocytes was discussed.The possibility that alpha hemolysin is a specific proteolytic enzyme is suggested by the observation that under no conditions could a separation be effected between the hemolytic and proteolytic properties of the alpha hemolysin recovered from a number of different cultures.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

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.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa
Keyword(s):  
Ion Exchange ◽  
Mixed Mode ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Charged State ◽  
Partial Structure ◽  
Reverse Phase ◽  
Oligomer Formation ◽  
Structure Rearrangement ◽  
Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

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