Purification and Properties of Three Pig Erythrocyte Carronic Anhydrases

Abstract

Three distinct forms of the zinc containing enzyme carbonic anhydrase were isolated from pig erythrocytes. One low activity type enzyme and two genetic variants of the high activity type enzyme with identical CO₂ hydratase activities which were 8 times as high were isolated from Danish Black and White Swine. In the isolation procedure described, the hemoglobin was eliminated by precipitation with chloroform-ethanol, and the isoenzymes were separated by DEAE-Sephadex chromatography. A number of enzymatically active minor components were separated. They were apparently all genetically linked to one of the three major components. The three purified isoenzymes behaved as homogeneous components during isoelectric focusing and electrophoresis at different pH values. They were characterized in terms of molecular weight, isoelectric pH, zinc content, amino acid composition, and enzymatic activity against CO₂, p-nitrophenyl acetate, and β-napthyl acetate. The circular dichroism of the enzymes in the ultraviolet region was studied. The properties of the enzymes were similar to those of carbonic anhydrases of corresponding types isolated from other mammalian species. Sulphur containing amino acid residues were absent in the low activity type enzyme. The amino acid composition of the two high activity mutants deviated only in that an arginine residue in the most widespread genetic variant was replaced by a histidine residue in the less frequent variant. Otherwise the two mutants showed identical properties.

Sammendrag

Tre distinkte former af det zinkholdige enzym carboanhydrase blev isoleret fra svineerytrocyter. Som fundet hos andre pattedyr indeholder svinets erytrocyter to typer carboanhydrase isoenzymer med forskellige kemiske, fysiske og katalytiske egenskaber. Et lavaktivt enzym med et isoelektrisk pH på 7,15 og et højaktivt enzym med et isoelektrisk pH på 5,97 og en 8 gange højere CO₂-hydrataseaktivitet isoleredes fra svin af Dansk Landrace. Fra danske sortbrogede svin kunne yderligere isoleres en genetisk variant af det højaktive enzym.

Isoleringsproceduren omfattede en fældning af hæmoglobinet med chloroform-ethanol og separation af isoenzymerne ved DEAE-Sephadex chromatografi. De tre enzymer opførte sig som homogene proteiner ved isoelektrisk focusering og elektroforese ved forskellige pH-værdier. Det lavaktive enzym udgjorde en fjerdedel af det aktive protein i alle chloroform-ethanol ekstrakter. Under fraktioneringen observeredes et stort antal enzymatisk aktive „minor components“, som tilsyneladende alle var genetisk koblet med en af de tre hovedkomponenter.

De tre oprensede isoenzymer blev karakteriseret med hensyn til molekylvægt, zinkindhold, aminosyresammensætning, ekstinktionskoefficient og enzymatisk aktivitet overfor CO₂, p-nitrophenylacetat og β-naphthylacetat. Enzymernes cirkular dichroisme blev også studeret. For de fleste af de studerede egenskabers vedkommende konstateredes tydelig lighed med isoenzymer af tilsvarende type isoleret fra andre pattedyr. Det lavaktive enzym indeholdt ingen svovlholdige aminosyrer. De to højaktive mutanter udviste næsten identisk aminosyresammensætning. Den eneste forskel, som blev observeret, var, at en argininrest i den mest udbredte variant var udskiftet med en histidinrest i den sjældnere variant. Bortset herfra konstateredes ingen signifikante forskelle i de to mutanters egenskaber.