Bovine carbonic anhydrase B

Fig. 38. Far-UV CD spectra of bovine carbonic anhydrase B in various states. Native,...

Fig. 39. Near-UV CD spectrum of bovine carbonic anhydrase B. Native state, pH 7 (—) acid-denatured state, pH 2 (---). See note in legend to Fig. 38. From Wong and Hamlin (1974). Biochemistry 13, 2678-2683, with permission. 1974, American Chemical Society.

Table 3. Metal ion specificity of bovine carbonic anhydrase B...

Figure 1 Refolding and aggregation model for bovine carbonic anhydrase B (CAB). When denatured CAB in 5 M GuHCl is rapidly diluted to refolding conditions, the unfolded protein, U, forms the first intermediate, 1 This intermediate continues to refold to the second intermediate, L, which proceeds to fold to the native protein, N. If the unfolded protein is diluted to aggregating conditions, the first intermediate will associate to form multimers. The dimer, D, and trimer, T, species can further associate, resulting in the formation of large aggregates. (Reproduced from ref. 1. Copyright 1990 American Chemical Society.)...

Wang, T., and Ikai, A., Protein stretching III. Force-extension curves of tethered bovine carbonic anhydrase B to the silicon substrate under native, intermediate and denaturing conditions, Jpn. J. Appl. Phys., 38, 3912-3917 (1999). 

Figure 1. NMR spectra of D2O solution of acetaldehyde In the presence of 1.3xl0 M bovine carbonic anhydrase B In 0.02 M phosphate buffer, pH meter reading 7.5. The total concentration of the acetaldehyde and Its hydrate was 0.46 M. The tenqperature was 28 0.

In an attempt to decide between these two possibilities, Lanir et al. have investigated the relaxation rates of the exchangeable water molecules in the inner coordination sphere of the metal in manganese-substituted bovine carbonic anhydrase B. Their conclusion is that one water molecule (and not a hydroxide ion) is directly bound to the metal at high pH values while below the pKa there is no rapidly exchanging water bound to the metal ion (cf. the conclusions of Koenig and Brown based on the results of similar studies with the cobalt enzyme). 

Chu, Y.H., Chen, J.K., Whitesides, G.M. (1993) Affinity Electrophoresis Multisectional Polyacrylamide Slab Gels Is a Useful and Convenien Technique for Measuring Binding Constants of Aiyl Sulfonamides to Bovine Carbonic Anhydrase B, Analytical Chemistry, Vol. 65, pp. 1314-1322. 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

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 B3.1.3 An isoelectric focusing (IEF) gel, pH 3 to 10. Lane 1, 4 pg purified egg white cystatin. Lane M, broad-range pi standards trypsinogen (pi 9.3), lentil lectin-basic band (pi 8.65), lentil lectin-middle band (pi 8.45), lentil lectin-acidic band (pi 8.15), myoglobin-basic band (pi 7.35 visible as a broad band), myoglobin-acidic band (pi 6.85), human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase (pi 5.85), a-lactoglobulin A (pi 5.20), soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50) in order shown from top of gel. The pi values of the two purified egg white cystatin isomers were determined to be 6.6 (upper band) and 5.8 (lower band). Adapted from Akpinar (1998) with permission from author.

The enzyme also catalyses the hydrolysis of various esters such as 4-nitrophenyl acetate and sultones. A number of slightly different enzymes, carbonic anhydrases A, B and C occur in different organisms. The most well characterised enzymes are the bovine and human carbonic anhydrases B, which are monomeric and contain one tightly bound zinc per 30000 molecular weight. 

Cd NMR spectra of Cd-substituted bovine carbonic anhydrase II in the presence of N-enriched (A) or N-enriched (B) benzenesulfonamide inhibitor. ... 

Figure 8. Electropherogram of six protein standards obtained by capillary zone electrophoresis. The standards are A sperm whale myoglobin B horse myoglobin C human carbonic anhydrase D bovine carbonic anhydrase E 13-lactoglobulin B ...

The hydration of CO2 occurs at a relatively slow rate in the absence of a catalyst, with a rate constant of 0.037 s at 25 °C (equation 12). The enzyme catalyzes the reaction by a factor of lO at pH 7, the human isoenzyme C being more active than human carbonic anhydrase B by a factor of 30. Bovine B enzyme is also of high activity. The value for binding CO2 is nearly independent... 

N.m.r. has been used to study the active site of other zinc-containing enzymes. In bovine carbonic anhydrase there is a single zinc co-ordination site available for Cl interaction, and the latter can be inhibited by CN and acetazolamide. C1 Resonance has also been used to investigate the environmental diflFerences of the zinc in carbonic anhydrase isozymes and the activation of pyruvate kinase with zinc, and to determine the pJ values in cobalt(u)-carbonic anhydrase. It is suggested that, although the TpK of aquozinc is about 9, the environment of the zinc ion in carbonic anhydrase B greatly increases the tendency of a zinc-bound water molecule... 

Cl NMR studies have been performed with human, horse and bovine carbonic anhydrases. Of these, the human and horse enzymes have been shown to exist in two principal forms differing in many properties and especially in the enzymatic activity. The forms of high specific activity are denoted C while the other major fractions of carbonic anhydrase are designed by the letter B. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Fig. 3 SDS-PAGE Photograph Separation (Lane Mr and A) and activity staining (Lane B and C) of the crude filtrate of Funalia trogii. Lane Mr standard molecular weight markers ([3-galactosi-dase, 118.0 kDa bovine serum albumin, 79.0 kDa ovalbumin, 47.0 kDa carbonic anhydrase, 33.0 kDa P-lactoglobulin, 25.0 kDa and lysozyme, 19.5 kDa). Relative mobilities of the standard markers vs. common logarithms of their molecular masses were plotted.With the linear regression output, the molecular masses of the proteins in the crude filtrate were estimated (taken from [18])...

Figure 3.9. SDS gel electrophoresis of MFGM proteins prepared by different washing procedures. S reference proteins phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, a-lactal-bumin. (Courtesy of J. Basch and H. M. Farrell, Jr.)...

To test this idea, a Bio-Rad Protean II electrophoresis cell and Bio-Rad Model 3000xi computer-controlled power supply were used to carry out the electrophoretic separation and recovery of pre-stained and unstained protein calibration standards (lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase B) obtained from Bio-Rad Laboratories. Standard SDS-gel electrophoresis techniques were used [167]. 

The most studied carbonic anhydrase is the one isolated from bovine or human red blood cells. It contains one zinc per 30 000 molecular weight, and exists as two major isoenzymes, B and C. Each isoenzyme has a distinct amino acid composition with about 60% sequence homology. 

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