Using Assays for Carbonic Anhydrase Activity and Inhibitor Screening

Carbonic anhydrase (CA) is a zinc-based metalloprotease found in at least 14 various isoforms in mammals. It catalyzes the reversible hydration of carbon dioxide (CO2) to produce proton and bicarbonate, thus playing a major role in pH and CO2 homeostasis. Alterations in CA activity are linked to various diseases such as type II diabetes mellitus, glaucoma, liver diseases, lung diseases, and so on.

Since CA is also a main target for cancer therapy, sensitive, robust, and high-throughput carbonic anhydrase activity and inhibitor screening assays are vital tools.

Carbonic anhydrase activity or carbon dioxide hydration rate is usually measured with the help of a potentiometer or a stopped-flow instrument as indicated by the change in pH over time. However, this technique is not appropriate for high-throughput assay. As a result, BioVision Inc. has launched the first high-throughput Carbonic Anhydrase Activity Assay Kit, which detects the esterase activity of this enzyme. This kit makes use of the zinc-hydroxide mechanism for carbon dioxide hydration and hydrolysis of an ester substrate leading to the release of a chromophore signal that is proportional to the enzyme activity.

CA Hydration and Esterase Activity

Catalytic cycle for human CA II catalyzed: (a) hydration of CO2 to HCO3 and H+ and (b) ester hydrolysis to carboxylic acid and alkoxide.

Figure 1. Catalytic cycle for human CA II catalyzed: (a) hydration of CO2 to HCO3 and H+ and (b) ester hydrolysis to carboxylic acid and alkoxide.

CA Assay Scheme

CA Assay Scheme

A Plate-Based Spectrophotometric Assay to Measure CA Activity with Intra- and Inter-Assay Variability

(A) Nitrophenol-standard curve (8–40 nmole), error bars indicate SD (n = 3). (B) Kinetic activity curves using different amounts of CA positive control in the assay.

Figure 2. (A) Nitrophenol-standard curve (8–40 nmole), error bars indicate SD (n = 3). (B) Kinetic activity curves using different amounts of CA positive control in the assay.

CA activity in mU per ng: (C) Intra-assay variation and coefficient of variation (n = 3). (D) Inter-assay variation and coefficient of variation (n = 3).

CA activity in mU per ng: (C) Intra-assay variation and coefficient of variation (n = 3). (D) Inter-assay variation and coefficient of variation (n = 3).

Application of Plate-Based CA Activity Assay Kit

(A) CA activity in hemolysate: Difference between the activity in absence and presence of inhibitor accounts for the Specific CA activity in hemolysate. (B) CA activity in serum: Difference between the activity in absence and presence of inhibitor accounts for the specific CA Activity in serum. (C) Specific CA activity: in hemolysate and serum were measured after 10x sample dilution with 2 µl of the diluted samples used in the assay.

Figure 3. (A) CA activity in hemolysate: Difference between the activity in absence and presence of inhibitor accounts for the Specific CA activity in hemolysate. (B) CA activity in serum: Difference between the activity in absence and presence of inhibitor accounts for the specific CA Activity in serum. (C) Specific CA activity: in hemolysate and serum were measured after 10x sample dilution with 2 µl of the diluted samples used in the assay.

Spike Recovery CA Activity in Hemolysate and Serum

Spike recovery of CA activity in serum and hemolysate: Samples were spiked with known amounts of CA and measured using BioVision’s CA Activity Assay Kit. The recovery was always ~100%.

Figure 4. Spike recovery of CA activity in serum and hemolysate: Samples were spiked with known amounts of CA and measured using BioVision’s CA Activity Assay Kit. The recovery was always ~100%.

IC50 Determined by BioVision’s CA Inhibition Screening Kit

Inhibition of CA activity by CA inhibitor (acetazolamide), IC50 = 16.3 ± 2 nM (n = 3). Assay was performed following the kit protocol.

Figure 5. Inhibition of CA activity by CA inhibitor (acetazolamide), IC50 = 16.3 ± 2 nM (n = 3). Assay was performed following the kit protocol.

Key Advantages of Carbonic Anhydrase Activity Assay Kit

  • Specific inhibitor is provided to measure specific enzyme activity
  • High-throughput homogenous assay with simple protocol: Direct spectrophotometric assay
  • Accurate reliable results: Reproducible results, 100% recovery of activity in spiking experiment
  • Stable reagents
  • Versatile sample applications

Applications

  • Measurement of activity of purified CA
  • Screening/characterizing ligands/inhibitors of CA
  • Measurement of CA activity in serum and hemolysate

Conclusions

BioVision’s Carbonic Anhydrase Activity and Inhibitor Screening Assays offer important tools for drug discovery and identifying potential modulators of CA Activity in a high-throughput format.

BioVision Incorporated

BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.

BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.


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Last updated: Feb 21, 2020 at 9:30 AM

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