Fish Physiology and Biochemistry vol. 12 no. 5 pp 381-386 (1994) Kugler Publications, Amsterdam/New York
The carbonic anhydrase inhibitor in trout plasma: purification and its effect on carbonic anhydrase activity and the Root effect Kenth Dimberg Department of Zoophysiology, Uppsala University, Norbyvagen 18 A, S-752 36, Uppsala, Sweden Accepted: September 28, 1993 Keywords: plasma inhibitor, purification, blood and gill carbonic anhydrase, Root effect
Abstract The plasma inhibitor of carbonic anhydrase (CA) from rainbow trout was purified using ion exchange chromatography. The inhibitor has a high isoelectric point value (pl > 10). SDS-PAGE electrophoresis and gel filtration demonstrated that the inhibitor is a low molecular weight compound of about 6,000 daltons. The plasma inhibitor was more effective against gill CA than against blood CA in vitro, probably reflecting the presence of various CA-isoenzymes in red blood cells and gill tissue. The apparent Root effect, i.e., the impairment of the oxygen binding capacity of hemoglobin in red blood associated with increased blood Pco 2 was counteracted by the plasma inhibitor, probably by acting on membrane-bound and/or cytosolic blood CA. This interaction may be of importance in adaptive mechanisms, e.g., during the acidemic phase, when the fish is being acclimated to hypercapnic conditions.
Introduction As early as 1938, Booth observed that a factor present in pig blood plasma inhibited the in vitro activity of erythrocytic carbonic anhydrase (CA). Endogenous inhibitors of CA have also been demonstrated in the plasma of fish (Haswell et al. 1983; Heming and Watson 1986) and several mammals (Hill 1986). It has been suggested that the function of natural inhibitors of CA is probably to inactivate the enzyme released into the plasma as a result of the normal erythrocyte aging process (Booth 1938). Another function could be to act on capillary endothelial CA in various respiratory tissues (Haswell et al. 1983). Haswell et al. (1983), who studied the biochemical nature of the inhibitor partially purified from eel blood plasma, found it to be a heat- and trypsinlabile protein. Still, the inhibitor's structure, its ac-
tion on membrane bound CA as well as on intracellular enzyme in vivo, and its physiological role remain to be investigated. The aim of the present study was to isolate the inhibitor in fish blood plasma and to examine its action on CA prepared from fish erythrocytes and gill tissue. In addition, the inhibitor's ability to interfere with the Root effect, i.e., the impairment of oxygen-loading capacity associated with a decrease in the intracellular pH in the red blood cells, was investigated.
Materials and methods Animals and plasma preparation Rainbow trout (Oncorhynchus mykiss) (400- 500 g) from Ns fish hatchery on the Dalilven River,
382 were used as experimental animals. The fish were acclimated for at least 2 months to aerated, flowing Uppsala tap water (8- 100 C, pH 7.7-7.9) prior to use in the experiments. Blood was drawn with a sodium-heparinized syringe from undisturbed fish via a catheter implanted in the dorsal aorta (Soivio et al. 1975). Immediately after blood sampling the plasma was separated by centrifugation at 1000 x g for 10 min. Plasma was pooled from four fish, giving a total volume of 25 ml, and then dialysed overnight at 4C against 21 ethanolamine buffer (20 mM, pH 12.0).
Inhibitorpurification Dialysed plasma was applied to a 2 x 5 cm column packed with an anion exchange medium (Q-Sepharose Fast Flow, Pharmacia) connected to a FPLC system (Pharmacia) at 40 C and equilibrated with the same ethanolamine buffer. After washing the column with buffer (flow rate 5 ml/min, 10 ml fractions) the salt concentration in the buffer was increased stepwise to 75, 100, 125 and 200 mM NaCI. The inhibitory effects of 1) plasma fractions obtained from each step of the chromatographic separation and 2) the final purified inhibitor were examined using a sensitive pH-stat system modified from the method described by Hansen and Magid (1966). An autotitrator assembly (Radiometer, autoburette ABU1, titrator 11, pH-meter 26 and a glass electrode GK 2401c) was used to measure the 0.01 and rate of CO 2 evolved at a pH of 7.40 was passed ml/min) of N (330 2°C. A stream 2 through a soda lime, Mallinckrodt CO 2 absorber and then used to remove the CO 2 produced in the reaction vessel. Gill or blood CA, prepared as below, was mixed in 10 ml of phosphate buffer (80 parts 1/15 M Na2 HPO4, 20 parts 1/15 M KH2 PO4 containing 0.1 mM of disodium-EDTA), separately with 200 gl of each plasma fraction of the chromatographic separation. For comparison, gill and blood CA enzyme extracts were diluted with the phosphate buffer to give approximately the same enzyme activity. After 2 min of incubation, the reaction was started with 0.5 ml 0.4 M NaHCO 3 as substrate, and the rate of CO 2 (mol CO 2 min- l )
formed by dehydration of HCO 3 was taken as being equivalent to the rate of proton addition by autotitration with 50 mM H 2 SO 4. Duplicate measurements were made, and the uncatalyzed reaction, corresponding to 0.25 glmol CO 2 min-1, was substracted from the total value. Fractions eluted at 125 mM NaCI, which contained the inhibitory activity, were pooled (40 ml) and dialysed overnight at 4°C against 21 of 20 mM ethanolamine buffer, pH 12.0. The dialysed sample was further purified at 4° C using an anion exchange column (Mono Q HR 5/5, Pharmacia) connected to a FPLC system. The column was equilibrated with 20 mM ethanolamine buffer (pH 12.0). After adding the sample, a linear NaCl gradient was applied and continued until the NaCl concentration reached 150 mM in a 20 mM ethanolamine buffer at pH 12.0 (flow rate 1 ml/min, total buffer volume 120 ml, 2 ml fractions). Fractions containing inhibitory activity were pooled, dialysed for 48h at 40 C against 4 1distilled water and freeze-dried. The sample was stored at 4°C prior to further analysis.
Polyacrylamide gel electrophoresis and gel filtration Half the amount of the freeze-dried inhibitory fraction prepared from 25 ml plasma was used for purity determination by using SDS-PAGE electrophoresis with the Phast System (Pharmacia, LKB). Sample and molecular weight markers (MW marker kit 1860-101, Pharmacia, LKB) were prepared separately in 1 ml of 10 mM Tris/HCl, 1 mM disodium-EDTA, pH 8.0. Sodium dodecyl sulfate (SDS) was added to reach 2.5% and the preparations were heated at 100 0 C for 5 min. A 4 1 amount of each preparation was run on Phast Gel high density media (Pharmacia, LKB) at 15 0C, 500 V, 10.0 mA for 30 min. The gel was silver stained according to Heukeshoven and Dernick (1985). The remainder of the inhibitory fraction was loaded on a column (1.6 x 40 cm) packed with Ultrogel AcA 202 (IBF) with a fractionation range of 1,00015,000. The sample was eluted with phosphate buffer (50 mM, 0.15 M NaCl, pH 7.2) at a rate
383 9 ml h-1 . Cytochrome C, Ribonuclease A and Aprotinin were used as molecular weight markers. One milliliter fractions were collected, monitored at 280 nm and tested for inhibitory activity on gill CA by using the CA assay technique described earlier.
Blood CA preparation Blood CA was prepared from erythrocytes taken with a sodium-heparinized syringe via the ductus cuvieri from captured rainbow trout. A 10 ml amount of blood was washed twice with Cortland saline, pH 7.5 (Wolf 1963) and then centrifuged at 1000 x g for 5 min at 40 C. The resuspended blood cells were subjected to a chloroform-ethanol extraction (10 ml blood suspension + 10 ml 40% ethanol + 5 ml chloroform) for 30 min at 40 C. Following extraction and centrifugation at 1000 x g for 15 min, the CA extract was freeze-dried. Enzyme extract was dissolved in Tris-SO 4 buffer (50 mM, pH 8.0, 0.2 mM EDTA) and applied on a column packed with Sephadex G-25 (PD-10 column, Pharmacia-LKB) equilibrated with the Tris-SO4 buffer. The enzyme was further purified by affinity chromatography on sulphanilamide sepharose gel according to Wihlstrand and Wistrand (1980). The gel column (2 x 4 cm) was equilibrated with the Tris-SO 4 buffer. After application of blood CA extract, the column was washed with sodium phosphate buffer (0.1 M, pH 6.5, 0.2 mM EDTA) followed by 0.2 M Na 2SO 4 in the same buffer. CA was eluted with 0.5 M sodium perchlorate in the phosphate buffer. One peak of CA was collected and dialysed against 4 1 1/15 M phosphate buffer, pH 7.4, for 48h at 4C.
Gill CA preparation Gill CA was prepared from gills washed to remove all traces of blood in accordance with Dimberg (1988). Gill tissue (1 g wet weight) was homogenised at 4C in 10 ml Tris-SO 4 buffer (50 mM, pH 8.0, 0.2 mM EDTA) containing 1% Triton X-100. After centrifugation at 20,000 x g for 30 min, the supernatant was applied on a PD-10 column equilibrated
with the Tris-SO4 buffer. The enzyme was purified further as described above for blood CA.
Apparent Root effect The oxygen-binding capacity of hemoglobin was determined in vitro in venous blood samples exposed to plasma inhibitor as well as acetazolamide, a well-known CA inhibitor, and the control substance sulfisoxazole (Swenson et al. 1981). The freeze-dried inhibitor fraction isolated from 10 ml of plasma was dissolved in 5 ml of Cortland saline, pH 7.5 (Wolf 1963). Ten millimolar acetazolamide (Lederle) and sulfisoxazole (Sigma) were prepared in Cortland saline at pH 12 and then titrated with 2 M HCI to a final pH of 7.5. Cortland saline, sulfisoxazole, acetazolamide or plasma inhibitor were mixed with whole blood (0.5 ml blood + 0.1 ml of tested substance) on ice and allowed to stand for 15 min. Subsequently, the preparation was equilibrated for 10 min at 10°C in a tonometer (Radiometer, AMT-1) supplied with two gases in the proportions 1.10% CO2, 17.0% 02 or 3.79% CO 2, 17.2% 02 with N2 as the residual gas. The amount of 02 in 100 tl blood was measured using a polarographic technique according to Solymar et al. (1971). The hemoglobin content was determined according to ICSH (1965), and the 02 content was expressed as the number of moles O2/mole of hemoglobin. Since the Root effect is affected directly by changes in intracellular pH, but only indirectly by the Pco, the impairment of the oxygen binding capacity of the hemoglobin associated with an increase in CO 2 content is referred to here as the apparent Root effect. The apparent Root effect (Re%) was expressed according to the following formula:
Re(%) - HbO2 (I) - HbO2 (II) Re(%) = HbO2 (I)
x 100
where HbO2(I) is the 02 content of the hemoglobin at low Pco and HbO 2(II) is the 02 content of the hemoglobin at high Pco02 Eight fish were used, and duplicate measurements were made. Student's t-test (paired samples) was used in the statistical analyses.
-AbSZBO
0
1 0
nm
,1 0-
2 0
3 0
-
40
CA
activity
5 0
6 0
Fraction number
Fig. I. Elution profile of the plasma inhibitor obtained after the final purification step on an anion exchange column (Mono Q, HR 5/5, Pharmacia). After sample application, a linear NaCl gradient was applied until the NaCl concentration reached 150 mM in a 20 mM ethanolamine buffer, pH 12.0, total buffer volume 120 ml. The flow rate was 1 ml/min, and 2 ml fractions were collected. Fractions were monitored at 280 nm and their inhibitory effect on gill carbonic anhydrase activity was measured in vitro.
Results and discussion Inhibitor purification Preliminary experiments showed that no plasma inhibitor adhered to the anion exchange media below pH 10, indicating that the inhibitory factor has a high isoelectric point (PI). In the final chromatographic step on the anionic exchange media run at pH 12 the plasma inhibitor eluted at a NaCl concentration of about 120 mM NaCl (Fig. 1). The purity of the pooled inhibitory fractions was assessed by SDS-polyacrylamide gel electrophoresis. The result of a typical gel electrophoretic run is shown in Figure 2. Purified plasma inhibitor migrated as a band with a molecular weight of 5,400 as compared with protein standards. Upon gel filtration with Ultrogel AcA-202 two protein peaks were observed. The first peak contained the inhibitor, which had a molecular weight of 6,900. The second peak had no inhibitory action, and its elution time indicated that it had a molecular weight of about 2,400. To estimate the molecular weight of small proteins using electrophoresis is a delicate task. Since the influence of the amino acid composition on peptide
Fig. 2. SDS polyacrylanide gel electrophoresis of protein molecular weight markers and purified pooled fractions (no 50-53) containing inhibitor from the anion exchange chromatographic step. The gel was run at 15OC, 500 V, 10.0 mA for 30 min, whereupon it was stained with silver. A = protein molecular weight markers (MW marker kit 1860-101, Pharmacia, LKB). B = pooled plasma inhibitor fractions.
properties is greater for shorter peptides than for longer ones, the small proteins do not necessarily follow the rules determined empirically and theoretically for estimating larger ones. The actual SDSpolyacrylamide gel is optimized for separation in the molecular weight range of 1,000-20,000 daltons; however, the molecular weight determination based on the electrophoretic technique should be considered as an apparent determination. Still, the results from the gel electrophoresis demonstrate that the chromatographic method applied in this investigation is suitable for use in purifying and isolating the plasma inhibitor. By using gel filtration technique Haswell et al. (1983) found that the eel plasma inhibitor is a low-molecular-weight protein between 10,000 and 30,000 daltons. Based on gel filtration, the present study revealed that the inhibitor in trout plasma is even smaller. It is entirely possible that the nature of plasma CA inhibitors differs between rainbow trout and eel. However, a more careful comparative investigation using the same chromatographic techniques is required to confirm that such a difference exists.
Inhibition of blood and gill CA activities The effects of purified plasma inhibitor on blood and gill CA are demonstrated in Figure 3. Evident-
385 I:
.
Gill-CA .0
E 5
x
0 oO
3 0
20 40 60 80 Inhibitor suspension ()
Fig. 3. Effects of increased amounts of plasma inhibitor on blood and gill carbonic anhydrase activities. Plasma inhibitor was prepared from 10 ml plasma and dissolved in 5 ml of the phosphate buffer used in the enzyme assay for measuring CA activity. Means + SD, duplicate measurements. I1 Intact blood [ Sulfisoxazole
El ·
Acetazolamide Plasma inhibitor
0 o
Fig. 4. Effects on the apparent Root effect associated with increased PCO2 in vitro in intact blood and in blood after being mixed with sulfisoxazole (control), acetazolamide or purified plasma inhibitor. Asterisks denote significant differences from control values (** p
The carbonic anhydrase inhibitor in trout plasma: purification and its effect on carbonic anhydrase activity and the Root effect Kenth Dimberg Department of Zoophysiology, Uppsala University, Norbyvagen 18 A, S-752 36, Uppsala, Sweden Accepted: September 28, 1993 Keywords: plasma inhibitor, purification, blood and gill carbonic anhydrase, Root effect
Abstract The plasma inhibitor of carbonic anhydrase (CA) from rainbow trout was purified using ion exchange chromatography. The inhibitor has a high isoelectric point value (pl > 10). SDS-PAGE electrophoresis and gel filtration demonstrated that the inhibitor is a low molecular weight compound of about 6,000 daltons. The plasma inhibitor was more effective against gill CA than against blood CA in vitro, probably reflecting the presence of various CA-isoenzymes in red blood cells and gill tissue. The apparent Root effect, i.e., the impairment of the oxygen binding capacity of hemoglobin in red blood associated with increased blood Pco 2 was counteracted by the plasma inhibitor, probably by acting on membrane-bound and/or cytosolic blood CA. This interaction may be of importance in adaptive mechanisms, e.g., during the acidemic phase, when the fish is being acclimated to hypercapnic conditions.
Introduction As early as 1938, Booth observed that a factor present in pig blood plasma inhibited the in vitro activity of erythrocytic carbonic anhydrase (CA). Endogenous inhibitors of CA have also been demonstrated in the plasma of fish (Haswell et al. 1983; Heming and Watson 1986) and several mammals (Hill 1986). It has been suggested that the function of natural inhibitors of CA is probably to inactivate the enzyme released into the plasma as a result of the normal erythrocyte aging process (Booth 1938). Another function could be to act on capillary endothelial CA in various respiratory tissues (Haswell et al. 1983). Haswell et al. (1983), who studied the biochemical nature of the inhibitor partially purified from eel blood plasma, found it to be a heat- and trypsinlabile protein. Still, the inhibitor's structure, its ac-
tion on membrane bound CA as well as on intracellular enzyme in vivo, and its physiological role remain to be investigated. The aim of the present study was to isolate the inhibitor in fish blood plasma and to examine its action on CA prepared from fish erythrocytes and gill tissue. In addition, the inhibitor's ability to interfere with the Root effect, i.e., the impairment of oxygen-loading capacity associated with a decrease in the intracellular pH in the red blood cells, was investigated.
Materials and methods Animals and plasma preparation Rainbow trout (Oncorhynchus mykiss) (400- 500 g) from Ns fish hatchery on the Dalilven River,
382 were used as experimental animals. The fish were acclimated for at least 2 months to aerated, flowing Uppsala tap water (8- 100 C, pH 7.7-7.9) prior to use in the experiments. Blood was drawn with a sodium-heparinized syringe from undisturbed fish via a catheter implanted in the dorsal aorta (Soivio et al. 1975). Immediately after blood sampling the plasma was separated by centrifugation at 1000 x g for 10 min. Plasma was pooled from four fish, giving a total volume of 25 ml, and then dialysed overnight at 4C against 21 ethanolamine buffer (20 mM, pH 12.0).
Inhibitorpurification Dialysed plasma was applied to a 2 x 5 cm column packed with an anion exchange medium (Q-Sepharose Fast Flow, Pharmacia) connected to a FPLC system (Pharmacia) at 40 C and equilibrated with the same ethanolamine buffer. After washing the column with buffer (flow rate 5 ml/min, 10 ml fractions) the salt concentration in the buffer was increased stepwise to 75, 100, 125 and 200 mM NaCI. The inhibitory effects of 1) plasma fractions obtained from each step of the chromatographic separation and 2) the final purified inhibitor were examined using a sensitive pH-stat system modified from the method described by Hansen and Magid (1966). An autotitrator assembly (Radiometer, autoburette ABU1, titrator 11, pH-meter 26 and a glass electrode GK 2401c) was used to measure the 0.01 and rate of CO 2 evolved at a pH of 7.40 was passed ml/min) of N (330 2°C. A stream 2 through a soda lime, Mallinckrodt CO 2 absorber and then used to remove the CO 2 produced in the reaction vessel. Gill or blood CA, prepared as below, was mixed in 10 ml of phosphate buffer (80 parts 1/15 M Na2 HPO4, 20 parts 1/15 M KH2 PO4 containing 0.1 mM of disodium-EDTA), separately with 200 gl of each plasma fraction of the chromatographic separation. For comparison, gill and blood CA enzyme extracts were diluted with the phosphate buffer to give approximately the same enzyme activity. After 2 min of incubation, the reaction was started with 0.5 ml 0.4 M NaHCO 3 as substrate, and the rate of CO 2 (mol CO 2 min- l )
formed by dehydration of HCO 3 was taken as being equivalent to the rate of proton addition by autotitration with 50 mM H 2 SO 4. Duplicate measurements were made, and the uncatalyzed reaction, corresponding to 0.25 glmol CO 2 min-1, was substracted from the total value. Fractions eluted at 125 mM NaCI, which contained the inhibitory activity, were pooled (40 ml) and dialysed overnight at 4°C against 21 of 20 mM ethanolamine buffer, pH 12.0. The dialysed sample was further purified at 4° C using an anion exchange column (Mono Q HR 5/5, Pharmacia) connected to a FPLC system. The column was equilibrated with 20 mM ethanolamine buffer (pH 12.0). After adding the sample, a linear NaCl gradient was applied and continued until the NaCl concentration reached 150 mM in a 20 mM ethanolamine buffer at pH 12.0 (flow rate 1 ml/min, total buffer volume 120 ml, 2 ml fractions). Fractions containing inhibitory activity were pooled, dialysed for 48h at 40 C against 4 1distilled water and freeze-dried. The sample was stored at 4°C prior to further analysis.
Polyacrylamide gel electrophoresis and gel filtration Half the amount of the freeze-dried inhibitory fraction prepared from 25 ml plasma was used for purity determination by using SDS-PAGE electrophoresis with the Phast System (Pharmacia, LKB). Sample and molecular weight markers (MW marker kit 1860-101, Pharmacia, LKB) were prepared separately in 1 ml of 10 mM Tris/HCl, 1 mM disodium-EDTA, pH 8.0. Sodium dodecyl sulfate (SDS) was added to reach 2.5% and the preparations were heated at 100 0 C for 5 min. A 4 1 amount of each preparation was run on Phast Gel high density media (Pharmacia, LKB) at 15 0C, 500 V, 10.0 mA for 30 min. The gel was silver stained according to Heukeshoven and Dernick (1985). The remainder of the inhibitory fraction was loaded on a column (1.6 x 40 cm) packed with Ultrogel AcA 202 (IBF) with a fractionation range of 1,00015,000. The sample was eluted with phosphate buffer (50 mM, 0.15 M NaCl, pH 7.2) at a rate
383 9 ml h-1 . Cytochrome C, Ribonuclease A and Aprotinin were used as molecular weight markers. One milliliter fractions were collected, monitored at 280 nm and tested for inhibitory activity on gill CA by using the CA assay technique described earlier.
Blood CA preparation Blood CA was prepared from erythrocytes taken with a sodium-heparinized syringe via the ductus cuvieri from captured rainbow trout. A 10 ml amount of blood was washed twice with Cortland saline, pH 7.5 (Wolf 1963) and then centrifuged at 1000 x g for 5 min at 40 C. The resuspended blood cells were subjected to a chloroform-ethanol extraction (10 ml blood suspension + 10 ml 40% ethanol + 5 ml chloroform) for 30 min at 40 C. Following extraction and centrifugation at 1000 x g for 15 min, the CA extract was freeze-dried. Enzyme extract was dissolved in Tris-SO 4 buffer (50 mM, pH 8.0, 0.2 mM EDTA) and applied on a column packed with Sephadex G-25 (PD-10 column, Pharmacia-LKB) equilibrated with the Tris-SO4 buffer. The enzyme was further purified by affinity chromatography on sulphanilamide sepharose gel according to Wihlstrand and Wistrand (1980). The gel column (2 x 4 cm) was equilibrated with the Tris-SO 4 buffer. After application of blood CA extract, the column was washed with sodium phosphate buffer (0.1 M, pH 6.5, 0.2 mM EDTA) followed by 0.2 M Na 2SO 4 in the same buffer. CA was eluted with 0.5 M sodium perchlorate in the phosphate buffer. One peak of CA was collected and dialysed against 4 1 1/15 M phosphate buffer, pH 7.4, for 48h at 4C.
Gill CA preparation Gill CA was prepared from gills washed to remove all traces of blood in accordance with Dimberg (1988). Gill tissue (1 g wet weight) was homogenised at 4C in 10 ml Tris-SO 4 buffer (50 mM, pH 8.0, 0.2 mM EDTA) containing 1% Triton X-100. After centrifugation at 20,000 x g for 30 min, the supernatant was applied on a PD-10 column equilibrated
with the Tris-SO4 buffer. The enzyme was purified further as described above for blood CA.
Apparent Root effect The oxygen-binding capacity of hemoglobin was determined in vitro in venous blood samples exposed to plasma inhibitor as well as acetazolamide, a well-known CA inhibitor, and the control substance sulfisoxazole (Swenson et al. 1981). The freeze-dried inhibitor fraction isolated from 10 ml of plasma was dissolved in 5 ml of Cortland saline, pH 7.5 (Wolf 1963). Ten millimolar acetazolamide (Lederle) and sulfisoxazole (Sigma) were prepared in Cortland saline at pH 12 and then titrated with 2 M HCI to a final pH of 7.5. Cortland saline, sulfisoxazole, acetazolamide or plasma inhibitor were mixed with whole blood (0.5 ml blood + 0.1 ml of tested substance) on ice and allowed to stand for 15 min. Subsequently, the preparation was equilibrated for 10 min at 10°C in a tonometer (Radiometer, AMT-1) supplied with two gases in the proportions 1.10% CO2, 17.0% 02 or 3.79% CO 2, 17.2% 02 with N2 as the residual gas. The amount of 02 in 100 tl blood was measured using a polarographic technique according to Solymar et al. (1971). The hemoglobin content was determined according to ICSH (1965), and the 02 content was expressed as the number of moles O2/mole of hemoglobin. Since the Root effect is affected directly by changes in intracellular pH, but only indirectly by the Pco, the impairment of the oxygen binding capacity of the hemoglobin associated with an increase in CO 2 content is referred to here as the apparent Root effect. The apparent Root effect (Re%) was expressed according to the following formula:
Re(%) - HbO2 (I) - HbO2 (II) Re(%) = HbO2 (I)
x 100
where HbO2(I) is the 02 content of the hemoglobin at low Pco and HbO 2(II) is the 02 content of the hemoglobin at high Pco02 Eight fish were used, and duplicate measurements were made. Student's t-test (paired samples) was used in the statistical analyses.
-AbSZBO
0
1 0
nm
,1 0-
2 0
3 0
-
40
CA
activity
5 0
6 0
Fraction number
Fig. I. Elution profile of the plasma inhibitor obtained after the final purification step on an anion exchange column (Mono Q, HR 5/5, Pharmacia). After sample application, a linear NaCl gradient was applied until the NaCl concentration reached 150 mM in a 20 mM ethanolamine buffer, pH 12.0, total buffer volume 120 ml. The flow rate was 1 ml/min, and 2 ml fractions were collected. Fractions were monitored at 280 nm and their inhibitory effect on gill carbonic anhydrase activity was measured in vitro.
Results and discussion Inhibitor purification Preliminary experiments showed that no plasma inhibitor adhered to the anion exchange media below pH 10, indicating that the inhibitory factor has a high isoelectric point (PI). In the final chromatographic step on the anionic exchange media run at pH 12 the plasma inhibitor eluted at a NaCl concentration of about 120 mM NaCl (Fig. 1). The purity of the pooled inhibitory fractions was assessed by SDS-polyacrylamide gel electrophoresis. The result of a typical gel electrophoretic run is shown in Figure 2. Purified plasma inhibitor migrated as a band with a molecular weight of 5,400 as compared with protein standards. Upon gel filtration with Ultrogel AcA-202 two protein peaks were observed. The first peak contained the inhibitor, which had a molecular weight of 6,900. The second peak had no inhibitory action, and its elution time indicated that it had a molecular weight of about 2,400. To estimate the molecular weight of small proteins using electrophoresis is a delicate task. Since the influence of the amino acid composition on peptide
Fig. 2. SDS polyacrylanide gel electrophoresis of protein molecular weight markers and purified pooled fractions (no 50-53) containing inhibitor from the anion exchange chromatographic step. The gel was run at 15OC, 500 V, 10.0 mA for 30 min, whereupon it was stained with silver. A = protein molecular weight markers (MW marker kit 1860-101, Pharmacia, LKB). B = pooled plasma inhibitor fractions.
properties is greater for shorter peptides than for longer ones, the small proteins do not necessarily follow the rules determined empirically and theoretically for estimating larger ones. The actual SDSpolyacrylamide gel is optimized for separation in the molecular weight range of 1,000-20,000 daltons; however, the molecular weight determination based on the electrophoretic technique should be considered as an apparent determination. Still, the results from the gel electrophoresis demonstrate that the chromatographic method applied in this investigation is suitable for use in purifying and isolating the plasma inhibitor. By using gel filtration technique Haswell et al. (1983) found that the eel plasma inhibitor is a low-molecular-weight protein between 10,000 and 30,000 daltons. Based on gel filtration, the present study revealed that the inhibitor in trout plasma is even smaller. It is entirely possible that the nature of plasma CA inhibitors differs between rainbow trout and eel. However, a more careful comparative investigation using the same chromatographic techniques is required to confirm that such a difference exists.
Inhibition of blood and gill CA activities The effects of purified plasma inhibitor on blood and gill CA are demonstrated in Figure 3. Evident-
385 I:
.
Gill-CA .0
E 5
x
0 oO
3 0
20 40 60 80 Inhibitor suspension ()
Fig. 3. Effects of increased amounts of plasma inhibitor on blood and gill carbonic anhydrase activities. Plasma inhibitor was prepared from 10 ml plasma and dissolved in 5 ml of the phosphate buffer used in the enzyme assay for measuring CA activity. Means + SD, duplicate measurements. I1 Intact blood [ Sulfisoxazole
El ·
Acetazolamide Plasma inhibitor
0 o
Fig. 4. Effects on the apparent Root effect associated with increased PCO2 in vitro in intact blood and in blood after being mixed with sulfisoxazole (control), acetazolamide or purified plasma inhibitor. Asterisks denote significant differences from control values (** p
