Determination Of Carbonic Anhydrase Activity Research Articles

A Colorimetric CO2 Hydration Assay for Facile, Accurate, and Precise Determination of Carbonic Anhydrase Activity

A simple, reliable, and universal method is demanded for routine determination of carbonic anhydrase (CA) activity, overcoming the limitations of previous assays that are inaccurate, complicated, expensive, or limited to a specific enzyme family. The most widely used Wilbur–Anderson assay was modified to improve the speed, accuracy, and precision by employing a temperature controllable UV/Vis spectrophotometer and the pH indicator phenol red. The experimental setting, measurement, and data analysis were facile and straightforward. The assay was validated using a commercially available bovine CA, showing that the obtained activity was directly proportional to the amount of enzyme. The measured activity (2540 WAU mg−1) agreed well with the previously reported data. The comparison results with esterase assay showed that the CO2 hydration assay should not be substituted by the esterase assay in the measurement of CA activity. The simple and reliable colorimetric method can be widely adopted for the routine determination of CO2 hydration activity, substituting for the traditional Wilbur–Anderson assay.

Metal Chelators Ethylenediaminetetraacetic acid (EDTA) and N,N,N′,N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) Interfere with Carbonic Anhydrase (CA) Activity in Wilbur‐Anderson Assay (WAa)

In our effort to set up a laboratory for undergraduate students with the tentative title ”Studying enzymatic activity of Carbonic Anhydrase”, we successfully determined carbonic anhydrase activity and demonstrated its inhibition by Acetazolamide, a potent CA inhibitor, using the Wilbur‐Anderson assay (WAa) using 6 experimental set‐ups with a class of 24 students.WAa hinges upon determining the time T (in seconds) required for 4 mL of saturated with carbon dioxide (CO2) water to lower pH from 8.3 to 6.3 of the mixture when added to 6 mL of 0.02M Tris buffer at 0°C without (T0) and with enzyme (TE). Activity (A) is defined as A = 2*(T0/TE‐1) assay units (AU). In n = 16 blank trials, T0 = 75.0±10.7s (Mean±Standard deviation), and with 0.001mg of CA in n = 12 trials, TE = 50.2±8.1s, A = 0.99±0.66 AU (p < 0.001). When for a test we used EDTA, a carboxylic high‐affinity metal ion chelator, to inactivate this Zn2+‐containing enzyme, we unexpectedly encountered that in the presence of the sodium salt of EDTA (Na2EDTA): 1) T was significantly slowed in a concentration‐dependent manner: for 5µM, 50µM, and 500µM, T was 84.3±7.9s (n = 10, p < 0.05), 108.5±.6s (n = 7, p < 0.001), and 130.8±22.7s (n = 10, p < 0.001), respectively; and 2) unexpectedly, in the context of the previous finding, Na2EDTA significantly enhanced CA activity (TE = 38.5±4.0s, A = 1.84 AU, n = 10, p < 0.001) in the presence of 5µM Na2EDTA that was abrogated by 10µM Acetazolamide, a potent CA inhibitor (T = 138.3±10.4s, n = 7, p < 0.001). TPEN, a pyridinic non‐carboxylic metal ion chelator that is more potent in metal ion chelation that EDTA, significantly slowed T0 (99.5±15.9s, n = 4) similar to Na2EDTA, and significantly enhance CA activity (TE = 41.1±3.8, n = 4, p < 0.05) with equal potency.This indicates that principally chemically different metal ion chelators EDTA and TPEN interfere with the determination of CA activity in the context of WAa. Studies to explain these phenomena are underway with the hypotheses that the contaminating metal ions that catalyze CO2 hydration/dehydration without CA are responsible for the observed phenomena.

New Strategy for in Vitro Determination of Carbonic Anhydrase Activity from Analysis of Oxygen-18 Isotopes of CO2.

The oxygen-18 isotopic (18O) composition in CO2 provides an important insight into the variation of rate in isotopic fractionation reaction regulated by carbonic anydrase (CA) metalloenzyme. This work aims to employ an 18O-isotope ratio-based analytical method for quantitative estimation of CA activity in erythrocytes for clinical testing purposes. Here, a new method has been developed that contains the measurements of 18O/16O isotope ratios during oxygen-18 isotopic exchange between 12C16O16O and H218O of an in vitro biochemical reaction controlled by erythrocytes CA and estimation of enzymatic activity of CA from the isotopic composition of CO2. We studied the enrichments of 18O-isotope of CO2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, H218O, and cellular produced CO2 on this reaction, we performed the in vitro experiments in closed containers with variations of those parameters. Finally, we mimicked the exchange reaction at 5% [CO2], 5‰ [H218O], pH of 7.4, and temperature of 37 °C to create the physiological environment equivalent to that of the human body and monitored the exchange kinetics with variations of CA activities, and subsequently, we derived the quantitative relation between the 18O-isotope of CO2 and CA activity in erythrocytes. This assay may be applicable for rapid and simple quantification of carbonic anhydrase activity which is very important to prevent the carbonic-anhydrase-associated disorders in human.

Potentiometric determination of carbonic anhydrase activity in rabbit carotid bodies: Comparison among normoxic, hyperoxic and hypoxic animals

The catalytic activity of carbonic anhydrase (CA) contained in the glomus cells of mammalian carotid bodies has been determined in vitro by a potentiometric method. Experiments performed on whole rabbit carotid bodies have shown a very low variability, in terms of the overall CA activity, among organs belonging to different animals maintained in normoxic conditions. Repeated assays performed on each carotid body have shown a marked decrease of the overall CA activity after the first assay, thus suggesting the presence of at least two different forms of enzyme. Experiments performed on carotid bodies belonging to rabbits maintained in normal, hyperoxic and hypoxic conditions have shown that the overall CA activity follows the sequence: hypoxic > normoxic > hyperoxic, matching with the corresponding physiological activity of the carotid body.

Determination of carbonic anhydrase activity by a pCO 2 sensor

A potentiometric method capable of determining carbonic anhydrase (CA) activity in vitro and based on the use of a pCO 2 sensor is presented. By means of the procedure described here it is possible to follow the rate of CO 2 diffusion that takes place in a buffered solution of NaHCO 3 in either the presence or the absence of CA. All experimental parameters that affect the speed of HCO 3 − dehydration, as well as the speed of CO 2 diffusion, can be fixed and kept constant for the duration of every assay. The advantage of this method is that the overall dehydration plus diffusion process can be followed as it actually takes place in open thermodynamic systems far from equilibrium. The results obtained strongly confirm the hypothesis of a facilitating role of CA toward the rate of CO 2 diffusion.

Determination of carbonic anhydrase activity using infra-red gas analysis

A technique for the estimation of carbonic anhydrase (CA) using the infra-red-gas analyser (IRGA) has been developed. Instead of determining pressure changes in the dehydration reaction catalysed by the enzyme, the IRGA was used to determine the increase in carbon dioxide content in the enzymatic and non-enzymatically catalysed dehydration reaction. The reliability of this method was assessed using pure carbonic anhydrase. Although the method has not been compared with other methods, the known sensitivity of the IRGA (resolution of 0.1–1.0 μmol/mol) was exploited. The differences in CA activity of two finger millet ( Eleusine coracana) genotypes differing markedly in photosynthetic rate but showing similar leaf conductances was assessed.

Fish erythrocytes are bicarbonate permeable: problems with determining carbonic anhydrase activity using the modified boat technique.

Effects of foaming agents (blood plasma, bovine serum albumin, polyvinyl-pyrrolidinone) and defoaming agents (octanol, n-foam) on manometric determination of carbonic anhydrase activity in intact erythrocytes of fish were examined. Foaming agents abolished the activity of red cells. Defoaming agents increased the activity and negated any differences in carbonic anhydrase activity between whole blood (red cells in plasma) and "Cortland" blood (red cells in saline). It is concluded that effects of plasma on the carbonic anhydrase activity of intact erythrocytes of fish measured using the modified boat technique are largely nonspecific and related to reagent foaming during mixing, rather than to the action of a specific enzyme inhibitor.

Carbonic anhydrase in marine algae.

An electrometric system for determination of carbonic anhydrase activity was constructed. Enzyme activity was assayed in homogenates of marine macroscopic Chlorophyta, Rhodophyta, and Phaeophyta. Plants surveyed included Ulva expansa (Setchell) Setchell and Gardner, Codium fragile (Suringar) Hariot, Enteromorpha sp., Chaetomorpha torta (Farlow) McClatchie (Chlorophyta); Laurencia papillosa (Greville). Plocamium coccineum var. pacificum (Kylin) Dawson, Pterocladia capillacea (Gmelin) Bornet and Thuret. Gigartina armata J. Agardh (Rhodophyta); Eisenia arborea Areschoug and Macrocystis pyrifera (Linnaeus) C. A. Agardh (Phaeophyta). Activity was present in all algae; in the Phaeophyta this could be demonstrated only after dialysis. p-Chloromercuriphenylsulfonic acid (10(-4)m) decreased activity in 1 species, Plocamium; this inhibition could be almost completely overcome with the addition of 10(-3)m dithiothreitol. In 2 green and 2 red algae assayed for sensitivity to acetazolamide (Diamox), inhibition was complete at 10(-4)m concentration of inhibitor. Dithiothreitol at a concentration of 10(-3)m did not enhance activity in any of the homogenates, and was not necessary for enzyme expression.

An ultramicro method for determination of carbonic anhydrase activity

Based on the CO 2-Veronal-Indicator method of Roughton and Booth, an ultramicro method for the determination of carbonic anhydrase was developed. Carbonic anhydrase activity can be determined in tissue fragments with dry weights between 10 and 200 mμg or in 0.2–0.4 μl tissue homogenates.

On the Determination of Carbonic Anhydrase in Biological Material

The activity of carbon,ic anhydrase can be determined by three methods, manometric, electrometric and colorimetric. The manometric procedure (Roughton & Booth, 1938 and 1946) is considered to be the most reliable but technically difficult. Both the electrometric (Wilbur & Anderson 1948) and colorimetric (Philpot & Philpolt 1936 and Roughton & Booth 1946) methods are founded on the change of pH, when H,CO:, is farmed from CO, and H,O, implying an increase of the acidity. The enzyme activity is then calculated as the inverse value of the number of seconds required to obtain a certain of pH. It may therefore be remarked that the enzyme has to react during a continuous change of pH, which will give a continuous fall of its activity. It may also be observed that some indicators inhibit the enzyme. These disadvantages diminish the reliability of the electronic and colorimetric methods. In this paper a new modification for determination of carbonic anhydrase activity is presented. The pH of the solution is kept constant during the entire determination. A preliminary note on the method has previously been published (Holmgkd 1960). The reaction is performed in an automatic titrator. Similar methods have been described for other enzymes, for instance acetylcholinesterase. When CO, reacts to H,CO,, the appropriate quantity of NaOH for maintsnce of a constant pH is automatically and contiiiuously added.

MICRO-DETERMINATION OF CARBONIC ANHYDRASE ACTIVITY IN ANIMAL TISSUE1

A modified manometric micro-method for the determination of carbonic anhydrase activity, using a pure carbonic anhydrase preparation for the definition of unit enzyme activity, was examined and established as a sensitive, accurate and practical method suitable for biological studies. Estimations of the carbonic anhydrase activity in the uteri of some mammalian species indicate that this method is useful for further investigations of this enzyme.

Carbonic anhydrase: a spectrophotometric assay

1. 1. A spectrophotometric method for the determination of carbonic anhydrase activity has been described. In this method a recording Beckman spectrophotometer model DK-1 has been used. 2. 2. The use of bicarbonate, barbiturate, and Tris buffers has been evaluated; the Tris buffer was found to be superior to both bicarbonate and barbiturate buffers in our experimental conditions. 3. 3. The method has been compared to previously published methods, and its advantages have been discussed.

Determination of Carbonic Anhydrase Activity

IN a recent paper, Wilbur and Anderson1 have compared the electrometric and colorimetric methods of determining carbonic anhydrase activity. They have confirmed an observation first made by Kiese and Hastings2 that indicators can inhibit the enzyme and introduce a source of error into the Philpot and Brinkman techniques. They maintain, therefore, the superiority of the electrometric method. However, it must be pointed out that any procedure which is based on measurements of the time taken to bring about a given change in pH, in the presence and absence of the enzyme, is much less reliable than the original manometric method of Meldrum and Roughton3. Experiments carried out in this laboratory have indicated that the electrometric method of following the pH changes is unsatisfactory when different substances are being tested for activating effects. Some of the errors are almost certainly due to interference with the rapid establishment of equilibrium between the glass electrode and the medium. Besides, there is always the possibility that the effects of activators and inhibitors depend on the pH at any given instant. For example, in a 4-ml. solution containing 4·5 units of carbonic anhydrase (activity = 160 units per mgm.), bromothymol blue at a concentration of 2·5 mgm. per cent inhibits by more than 45 per cent at pH 6·8 but only by 25 per cent at pH 7·5. It is suggested, therefore, that the 'boat' method must remain the standard procedure, particularly in work dealing with activators and inhibitors.