Inhibitory effects of some phenolic compounds on the activities of carbonic anhydrase: from in vivo to ex vivo

Figures & data

Figure 1. The chemical structure of phenolic compounds, including arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2,4,6-trihydroxybenzaldehyde as inhibitors for hCA I and II isozymes.

Figure 2. Elution graph of the hCA I and II isoenzymes purified by using Sepharose-4B-L-Tyrosine affinity chromatography.

Figure 3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified CA I and II isozymes from human erythrocytes. Lane A and D: standard proteins (kD): β-galactosidase from E. coli (116), phosphorylase B from rabbit muscle (97), bovine serum albumin (66), chicken ovalbumin (45) and carbonic anhydrase (29). Lanes B and F: human carbonic anhydrase I. Lanes C and E: human carbonic anhydrase II.

Table 1. Summary scheme of hCA I and II isoenzymes purified using Sepharose-4B-L-tyrosine affinity chromatography.

Sample type	Activity (EU/mL)	Total volume (mL)	Protein (mg/mL)	Total protein (mg)	Total activity	Specific activity (EU/mg)	Yield (%)	Purification fold
Haemolysate	1020	77.5	78.98	6121.00	79 050	12.9	100	1
Sepharose-4B-L-tyrosine-sulphanilamide affinity column chromatography
CA I	5934	7.5	1.26	19.65	44 505	4709.5	56	365
C CA II	3096	3.5	0.28	0.98	10 836	11057.1	14	856

Table 2. The IC₅₀, K_i and inhibition type of phenolic compounds for the hCA I and II isozymes as ex vivo using the esterase activity method.

	hCA I			hCA II
Phenolic compounds	IC50 (μM)	Ki (μM)	Inhibition type	IC50 (μM)	Ki (μM)	Inhibition type
Arachidonoyl dopamine	346.50	203.80	Non-competitive	231.00	75.25	Uncompetitive
2,4,6-Trihydroxybenzaldehyde	1380.00	1170.00	Competitive	77.00	354.00	Non-competitive
3,4-Dihydroxy-5-methoxybenzoic acid	1550.00	910.00	Competitive	820.00	1510.00	Non-competitive

Table 3. The IC₅₀ values of substances for the hCA I and II isozymes ex vivo using the CO₂-hydratase activity method.

	hCA I		hCA II
Phenolic compounds	IC50 (μM)	r^2	IC50 (μM)	r^2
Arachidonoyl dopamine	173.25	0.9971	1070.00	0.9948
2,4,6-Trihydroxybenzaldehyde	115.50	0.9468	1360.00	0.9635
3,4-Dihydroxy-5-methoxybenzoic acid	542.88	0.9767	693.00	0.9991

Figure 4. The half-life (t₅₀) values of the substances were calculated by activity%-elapsed time graphs in in vivo studies. The half-life (t₅₀) values were found for arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2,4,6-trihydroxybenzaldehyde. These experiments were performed using CO₂-hydratase activity assays.

Table 4. The activity values of substances for in vivo rat erythrocyte CA enzyme as IU/gHb using the CO₂-hydratase activity method.

Groups*
Time (hour)	Control	Arachidonoyl dopamine	3,4-Dihydroxy- 5-methoxybenzoic acid	2,4,6-Trihydroxybenzaldeyde
First	54 927.1 ± 1258.8^Ψ^,^a	46 953.0 ± 730.0^ϕ^,^a	47 067.0 ± 1246.9^ϕ^,^a	45 698.8 ± 641.2^ϕ^,^a
Fourth	55 589.5 ± 1262.5^Ψ^,^a	40 661.6 ± 1750.7^ϕ^,^b	39 357.5 ± 930.3^ϕ^,^b	37 781.4 ± 1182.4^Ω^,^b

*CA activity as IU/gHb.

^Ψ,ϕ,Ωp < 0.05. The difference between the groups with different uppercase letters on the same line was significant.

^a,bp < 0.05. The difference between the time values with having different lowercase letters in the same column was significant (first and fourth hours).

Table 5. The half-life (t₅₀) values of substances for rat erythrocytes CA enzyme using the CO₂-hydratase activity method.

Phenolic compounds	Half-life (hour)
Arachidonoyl dopamine	8.34
3,4-Dihydroxy-5-methoxybenzoic acid	7.70
2,4,6-Trihydroxybenzaldeyde	6.79