Anhydrase-method

The interaction of carbon disulfide as a substrate in carbonic anhydrase model systems has been studied using density functional theory methods. A higher activation energy of CS2 compared to C02 in the reaction with [L3ZnOH]+ was due to the reduced electrophilicity of CS2. The reversibility of the reaction on the basis of these calculations is questionable with [L3ZnSC(0)SH]+ as intermediate.572... 

As examples. Table 8 records some observations on d—d and charge transfer absorption bands in metal/protein systems. The examination of the spectrum of cobalt carbonic anhydrase (d—d) and of iron conalbumin (charge-transfer) permitted a prediction of the ligands from the protein to the metal. The predictions have now been substantiated by other methods. 

A great deal of discussion about elemental determination methods focuses on minor, trace, ultra-trace levels of analyte presence in relatively large volume samples. There is another area equally challenging and that involves elemental determination at major and minor concentration in very small volume/low mass samples. Lochmuller and Galbraith used PIXE to study the metal content of carbonic anhydrase... 

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. ... 

Although P MRS can be used to measure pH in isolated, perfused hearts, it is not suitable in vivo because 2,3-diphosphoglycerate in ventricular blood interferes with the myocardial Pi resonance. Schroeder et al. have developed a possible alternative method for in vivo use based on the carbonic-anhydrase-catalyzed, pH-dependent equilibrium of CO2 and HCO3. Infusion of h)q)erpolarized 1- C-pyruvate generates strong signals of metabolically produced C02 and H COJ to yield pH by the Henderson-Hasselbalch equation. Applicability of the method in humans remains to be demonstrated. 

Applications to Biological Samples. - Methods of distance measurements were compared for four doubly spin-labelled derivatives of human carbonic anhydrase.53 The distances between the spin labels were obtained from continuous wave spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of the line-shape broadening, and computer simulation of line-shape changes. For variants with interspin distances greater than 18 A, the DEER method also was used. For each variant, at least two methods were applicable and reasonable agreement between distances obtained by different methods was obtained. The useful distance ranges for the techniques employed at X-band with natural isotope abundance spin labels were estimated to be half-field transition (5-10 A), line-shape simulation (up to 15 A), Fourier deconvolution (8 - 20 A), and four-pulse DEER (> 18 A).53... 

Carbon Dioxide. The U.S. limit for carbon dioxide in non-sparkling wine is 2.77 grams/liter. Above this value the tax rises from 170 per gallon to 2.40 or 3.40. Other countries have less stringent limits. Obviously an accurate method is required, and several are available (4, 5, 6). At present a simple enzymatic reaction using carbonic anhydrase is preferred. The bicarbonate ion is titrated with standard acid between pH 8.6 and 4.0. Carbonic anhydrase ensures that the carbonic acid is all in the bicarbonate form. A non-fading endpoint is thus obtained. 

The method has been applied widely using, for example, methylamine. A newer method employs an isotope exchange procedure to measure the pH-sensitive carbonic anhydrase activity naturally present in mitochondria.181... 

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.

It should be noted that the kinetics for the human B isoenzyme are more complicated in that the pH dependence indicates that additional groups influence the rate. Studies with the isoenzyme carboxymethylated at His-200 prepared from 13C-labelled bromoacetate show that the pH dependence of the 13C NMR signal can be fitted to a curve with two pKa values of 6.0 and 9.2, but not to a curve with a single pKa. The second group could be the imidazole side-chain of His-200. Paramagnetically shifted 13C NMR resonances in the modified Co11 human carbonic anhydrase-B have been located by a novel method 498 This should allow the confirmation of an earlier postulate that the carboxymethyl carboxylate is a ligand for zinc in the modified enzyme. 

The dimeric nature of alkaline phosphatase makes it a more complicated system than carbonic anhydrase or carboxypeptidase. The enzyme contains several metal-binding sites. The stoichiometry of zinc binding is not completely settled. There are at least two strongly bound metal ions (109, 111, 114), but the presence of four specific sites has been claimed (115, 116). At alkaline pH, the enzyme tends to bind even more zinc rather strongly, but probably to sites unrelated to catalytic function (109). A critical evaluation of this aspect falls outside the scope of this review, but it appears that some of the apparent discrepancies are due to different experimental methods in measuring metal binding. 

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

The importance of carbon dioxide in the formation of the iron complexes of the human serum transferrin was shown early by Fiala and Burk (46) and Schade et al. 117). Warner and Weber (133), by an indirect but exquisite method, proved the participation of CO2 in the formation of the complex, and also that it was in the form of bicarbonate or carbonate. This was done through experiments in which the iron complex was formed in the absence of CO2, or by the addition of small amounts, of gaseous CO2, and noting the red color of the complex was formed only very slowly. However, when the enzyme carbonic anhydrase was added, together with the CO2, the red color of the complex appeared rapidly. In addition the same workers showed by the use of C1402 that one mole of CO2 was bound per mole of iron bound. 

Persson, M., Harbridge, J. R., Hammerstrom, P., Mitri, R., Ma rtenson, L-G., Carlson, U., Eaton, G., and Eaton, S. (2001) Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II, Biophys. J. 80, 2886-2897. 

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