Carbonic anhydrase purification

Maren TH (1967) Carbonic anhydrase Chemistry, physiology, and inhibition. Physiol Rev 47 595-781 Philpot FJ, Philpot JSL (1936) A modified colorimetric estimation of carbonic anhydrase. Biochem J 30 2191-2193 Wistrand PJ, Knuuttila K-G (1980) Bovine lens carbonic anhydrases Purification and properties. Exp Eye Res 30 277-290... 

Keilin, D., Mann, J., Carbonic Anhydrase. Purification and Nature of... 

Different enzymes exhibit different specific activities and turnover numbers. The specific activity is a measure of enzyme purity and is defined as the number of enzyme units per milligram of protein. During the purification of an enzyme, the specific activity increases, and it reaches its maximum when the enzyme is in the pure state. The turnover number of an enzyme is the maximal number of moles of substrate hydrolyzed per mole of enzyme per unit time [63], For example, carbonic anhydrase, found in red blood cells, is a very active enzyme with a turnover number of 36 X 106/min per enzyme molecule. It catalyzes a very important reaction of reversible hydration of dissolved carbon dioxide in blood to form carbonic acid [57, p. 220],... 

Very recently the isolation of still active carbonic anhydrase from the biocrystalline layer of hens egg shells has been reported. Crude egg shell extracts exhibited no enzyme activity. however, after removal of Ca2+ and simple gel filtration of the extracts, carbonic anhydrase activity could be measured. Further purification steps led to the isolation of two isoenzymes. The possibility of inhibition of this enzyme activity by shell components cannot be excluded although recombination of shell components after separation did not inhibit that enzyme. The isolated enzymes has an apparent molecular size of 28,000 daltons. Carbonic anhydrase from egg shells catalyzes the reversible hydration of CO2 + HOH H+ + HCO3-. This probably is the primary action of the enzyme of the shell. Moreover, egg shell carbonic anhydrase catalyzes the hydration of acetaldehyde and pyridine aldehyde. Furthermore, the same enzymes have esterase activity (hydrolysis of p-nitrophenyl acetate). Whether the latter activities play a role in the egg shell cannot be judget at the present time. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

The measurement of activity, isolation and purification, activation, inhibition, and distribution of carbonic anhydrase have been reviewed (Van Goor, 1948 Vallee and Altschule, 1949 Roughton and Clark, 1951 Weier and Stocking, 1952). A summary of its physical and chemical properties will be given here. 

Since at the time at which this work was undertaken carbonic anhydrase was the only known zinc metalloenzyme (Keilin and Mann, 1940a), and no carbonic anhydrase activity could be detected in leukocytes (Van Goor, 1948), the work on the purification of the zinc-containing protein of leukocytes (Hoch and Vallee, 1952, 1953) was undertaken to elucidate the physiological and biochemical significance of zinc in human leukocytes through a study of its association with other components of these cells. 

Since all zinc of human erythrocytes is presumably structurally associated with carbonic anhydrase (Keilin and Mann, 1940a Vallee et al., 1949), the identification of this enzyme in subsequent fractionations provided an indirect check on these calculations. The only fraction in the process of purification exhibiting significant carbonic anhydrase activity was precipitated between 0.85 and 0.90 of full saturation with ammonium sulfate (Table IV). 

Carbonic anhydrase from Pisum sativum has been purified by affinity chromatography and SDS-PAGE. In Fig. 1, the series of purification steps are described and the polypeptide profiles of the active fractions are shown. For comparison, the polypeptides eluted from a section of a native PAG exhibiting CA activity are also shown. CA activity in all purification steps was found to be associated with a polypeptide doublet of 25.5 kDa and 27.5 kDa which is similar to other reported CA... 

Figure 1. Purification of pea carbonic anhydrase. Tb figure shows a Coomassie Blue stained, 7-15% lineaf gradient, SDS polyacrlyamide gel (PAG) loaded with total soluble protein (lane G), 30-60% (NH4)2S04 precipitate (lane F), eluate from the affinity columt chromatography (lane E) and after excision and electroelution from an SDS-PAG of the affinity column eluate (lane D). Lane C contains the polypeptides found in fractions exhibiting CA activity after Sephadex G-200 chromatography of the affinity column eluate and lane B contains polypeptides electroeluted from a section of a native PAG stained for CA activity. A Western blot of the total soluble protein (lane G) probed with the CA polyclonal antisera is shown in lane I. Molecular weight markers (Bio-Rad Inc.) are shown in lanes A and H...

Studies of the recycling of the cofactor in an enzyme reactor comparison of reactions using free or immobilized nicotinamide adenine dinucleotide Investigations of the role of carbonic anhydrases in vivo Purification of tryptophanase and tyrosine phenol-lyase... 

Ozensoy, O. Arslan, O. Sinan, O.S. (2004). A new method for the purification of carbonic anhydrase isozymes by affinity chromatography. Biochemistry (Moscow), 69, 216-219. 

Zhu, X.L. Sly, W.S. (1990). Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney. Journal of Biological Chemistry, 265, 8795-8801. 

Figure 3 Sodium dodecyi suifate/poiyacryiamide gel eiectrophoregram of the final purification steps was carried out in slab gels. The silver staining was used to reveal proteins in the separating gel (10 % polyacrylamide). Lane 1 Solubilized spherosomal membranes. Lane 2 Dialyzed fractions. Lane 3, 4 and 5 FPLC active fraction, lane 6 molecular weight markers (phosphorylase A, Mr 100 kDa bo 4n serum albumin, Mr 66 kDa carbonic anhydrase, Mr 30 kDa).

Resolution by metal chelate-type HPAC application to ribonuclease A, transferrin and carbonic anhydrase Resolution and purification by hydrophobic-type HPLC at a preparative scale... 

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