Carbonic anhydrase assay

DDT can be determined in microgram amounts by its inhibition of carbonic anhydrase (69). Several of the chlorinated insecticides as well as the carbamate insecticide, Sevin, are potent lipase inhibitors. As little as 0.1-1 /Ag/ml of Sevin, aldrin, heptachlor, lindane, 10 / g/ml of DDT, and 2,4-d can be determined fluorimetrically with 2-3% precision by the lipase inhibition when 4-methyl umbelliferone heptanoate is used as substrate (70). The lipase inhibition procedure for DDT determination is much easier to carry out than the carbonic anhydrase assay which involves monitoring gaseous COo production. 

Carbonic Anhydrase assay - CA activity was assayed according to the method of Rickli et al.(1964), with a certain modification. The reaction mixture contained 2.5 ml of 0.025 M KH2P0 buffer, pH 8.3 and phenol rot (2.5 mg.per 100 ml.). Enzyme solution 0.1 ml. of appropriate concentration (0.15-0.20 mg.for cytoplasmic CA and 0.06-0.10 mg.for stromal CA) was added and 2.5 ml of cold KHCO, was injected into the assay mixture. The rate of non-cataiyzed reaction was recorded from the moment of injection on KHCO, to a change in the colour of the indicator form yellow to" orange. The enzyme activity was calculated as difference in the seconds between the control and the catalitic reactions and was expressed as pM C0,min-1. mg.pr."1. 

Proobstational Activity by Endometrial Carbonic Anhydrase Assay of a Group op Prooestins... 

Carbonic anhydrase (CA) exists in three known soluble forms in humans. All three isozymes (CA I, CA II, and CA III) are monomeric, zinc metalloenzymes with a molecular weight of approximately 29,000. The enzymes catalyze the reaction for the reversible hydration of C02. The CA I deficiency is known to cause renal tubular acidosis and nerve deafness. Deficiency of CA II produces osteopetrosis, renal tubular acidosis, and cerebral calcification. More than 40 CA II-defi-cient patients with a wide variety of ethnic origins have been reported. Both syndromes are autosomal recessive disorders. Enzymatic confirmation can be made by quantitating the CA I and CA II levels in red blood cells. Normally, CA I and CAII each contribute about 50% of the total activity, and the CAI activity is completely abolished by the addition of sodium iodide in the assay system (S22). The cDNA and genomic DNA for human CA I and II have been isolated and sequenced (B34, M33, V9). Structural gene mutations, such as missense mutation, nonsense... 

A method for the assay (enzyme aetivity) of carbonic anhydrase (Chap. 8. Zn(ll)) uses the catalysis at pH 7.0 of the hydrolysis reaction... 

For reactions in which one or more reactants or products is a gas, manometry (the measurement of pressure differences) can provide a convenient means for monitoring the course and kinetics of the reaction Thus, enzymes that can be assayed with this method include oxidases, urease, carbonic anhydrase, hydrogenase, and decarboxylases. For example, bacterial glutamate decarboxylase is readily assayed by utilizing a Warburg flask and measuring the volume of gas evolved at different times using a constant-pressure respirometer. ... 

Fig. 7 Mobility-shift assay for the determination of dissociation constant of the complex between anti-DNP rat monoclonal IgG21) antibody and charged ligands that contained the A-dinitrophenyl group. Mesityl oxide (MO) served as EOF marker, bovine carbonic anhydrase (CAB) and bovine a-lactalbumin (LA) as internal references. The DNP ligands with a charge of —1 (A) und —9 (B), respectively, were used as additives to the running buffer. (Reprinted with permission from Ref. 30. Copyright 1995 American Chemical Society.)...

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.

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

Use of apoenzymes for the detection of metal ions Generally, apoenzymes of metalloenzymes can be used for the detection of the corresponding metal ion. Restoration of enzyme activity obtained in the presence of the metal ion can be correlated to its concentration. This principle has been demonstrated in the detection of copper while evaluating reconstituted catalytic activities in galactose oxidase and ascorbate oxidase and also in the detection of zinc since this ion is essential for the activity of carbonic anhydrase and alkaline phosphatase [416]. The need of stripping the metal for the preparation of the apoenz5une may demand tedious procedures and a catalytic assay with the addition of the substrate is always required for detection. 

The report by Haas and Guardia (18) pertains to their efforts to apply immunological methods for the assay of pesticide residues, and expresses their aim to test the suitability of methods for field analytical purposes. Haas and Guardia also used DDT and malathion to represent two of the most important classes of insecticides, chlorinated hydrocarbons and organophosphorus compounds. They first attempted to prepare insecticide-protein antigens in which enzymes were used as the protein carrier. Antiserum of rabbits injected with DDA-carbonic anhydrase or malathion-chymotrypsin failed to show the presence of the respective antibodies. 

Figure 8.15 Colourimetric assay. Carbonic Anhydrase (CA) assay system. Changes in pH with enzyme catalysis are followed with great precision and accuracy by pH-sensitive dye that changes absorbance profile with pH-change.

CA[Mn] efficiently catalyzed the oxidation of o-dianisidine to the red quinonedi-imine with hydrogen peroxide (Scheme 2). This o-dianisidine oxidation is a common assay for peroxidase [47-50] or for peroxidase-based detection of hydrogen peroxide [51]. As control reactions, native zinc carbonic anhydrase showed <1% of the activity of CA[Mn], while the same concentrations of manganese(ll) chloride and bicarbonate alone showed only 5% of the activity of CA[Mn]. Unlike other peroxidases [47-50], the CA[Mn]-catalyzed oxidation of o-dianisidine required bicarbonate and showed only 1.5% of the activity in the absence of bicarbonate. 

An interesting assay for progestational activity was developed by Lutwak-Mann [64, 65], who observed that the carbonic anhydrase concentration of... 

Dichlorodiphenyltrichloroethane (DDT) inhibits carbonic anhydrase at the amount of micrograms at which other inhibitors are inactive. Carbonic anhydrase catalyzes the hydration of carbon dioxide to carbonic acid. The activity of the enzyme is assayed by measuring the CO2 liberated by dehydration in a Warburg manometer or a CO2 gas-selective electrode. DDT inhibits the enzyme, causing a decrease in the rate of CO2 liberation. 

Fig. 1. Ultrogel AcA 54 chromatography of sARF I effect of dimyristoyl phosphatidylcholine (DMPC) on ADP-ribosylation of Gsa and choleragen A protein. Purified sARF I was chromatographed on a column (1.2 x 104 cm) of Ultrogel AcA 54. Fractions (1 ml) were collected and samples were (A) analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed for ARF activity in a reaction mixture containing Gg (0.4 jig), choleragen (25 pg), and 100 pM GTP without (B) or with (C) 1 mM DMPC. (A) SDS-PAGE of 250 pi of fraction plus bovine serum albumin, 10 pg. Lanes 1-4, fractions 68, 72, 76 and 80 Lane 5, standard proteins, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, a-lactalbumin. (B) and (C) Autoradiograms of ADP-ribosylated proteins. Lanes 1-4, fractions 68,72,76 and 80 Lane 5, column buffer.

Physiol Land 94 16P-17P, 1938-39.1 could assay carbonic anhydrase quickly, because my Balliol College tutor had with his wife devised a simple method for its rough measurement. Philpot FJ, Philpot JStL. A modified colorimetric estimation of carbonic anhydrase. Bio-c/zt m 7 30 2191-2193, 1936. 

Shingles, R. Moroney, J. V. Measurement of carbonic anhydrase activity using a sensitive fluorometric assay. Anal. Biochem. 1997, 252, 190-197. 

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