Comp. Biochem. Physiol.. 1976, Vol. 54B, pp. 331 to 332. Peroamon Press. Printed in Great Britail~
CATALASE IN FISH RED BLOOD CELLS A. C. SMIrd Department of Pathology, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822, U.S.A.
(Received 19 August 1975) Abstraet--Catalase activity was measured in the red blood cells from four different teleostean species: the milkfish, Chanos chanos; palani, Acanthurus dussumieri; naso, Naso lituratus; and butterfly fish, Chaetodon miliaris. 2. The enzyme activity and associated red cell color changes on exposure to air, correlated with the taxonomic relationships of the species studied. 3. Catalase was detected in all species except the most primitive--the milkfish--wh~, h revealed zero to minimal activity. These results are consistent with those of the only other known study (Rabie et al., 1972), on different species. 4. Catalase, therefore, may have evolved with the higher teleosts. 5. The milkfish is suggested as a possible model for a- or hypo-catalasemia in humans. INTRODUCTION The only report, to the author's knowledge, describing catalase investigations on the red blood cells of fishes is by Rabie et al. (1972). These authors worked with one of two living representatives of the primitive chondrosteans, Polypterus senegalus, and three modern teleosts (Tilapia nilotica, Clarias lazera, and Synodontis shall). Results indicated no catalase activity in the red cells of Polypterus, but definite, although variable, activity in the red cells of the other species. In Hawaiian waters, representatives of both primitive and modern teleostean fishes are available. Included in the former category is the milkfish, Chanos chanos. This species is intermediate in evolutionary position between the primitive and modern fishes of the study by Rabie et al. In the latter category are a great variety of reef fishes. These Hawaiian fishes could be a resource to extend studies of the taxonomic distribution of catalase, with its concomitant evolutionary implications, as begun by the authors mentioned. Further, the local availability of an a- or hypo-catalasemic fish would greatly facilitate medically oriented research underway on catalase in general. This work currently utilizes specially bred acatalasemic mice. MATERIALS A N D M E T H O D S
Fishes studied All fishes were collected in Hawaiian waters. Three individuals of each of the following species were used: milkfish, C. chanos; palani, Acanthurus dussumieri; naso, Naso lituratus; and butterfly fish, Chaetodon miliaris. Blood Sampling Blood samples were obtained by severing the caudal peduncle with a knife and collecting the blood from each species in a vial containing approx 0.2 ml of a super-saturated solution of the anticoagulant, EDTA*. The blood samples were centrifuged at approx 1171 g for five min at * Ethylenediaminetetracetic acid.
room temperature (25°C), and the supernatant plasma was discarded. The cells were then alternately washed with saline (0.85 g % NaC1) solution and centrifuged as before, 3-times. After the last centrifugation, the saline wash solution was discarded and the remaining blood cells were stored frozen until ready for testing.
Testing for Catalase (a) Subjective method. One drop of 3% hydrogen peroxide was added to one drop of the thawed blood (red) cell preparation on a glass slide. The reaction, in terms of amount of bubbling, was ranked 0 (no reaction) to 4 + (maximum bubbling, equal to that produced when normal human red cells are used). Red cells pooled from acatalasemic mice and normal humans served as controls. In addition, observations were made on the thawed red cells exposed to the air for a period of up to approx 2½ hr. The appearance of any color changes was noted. (b) Objective method. This procedure was carried out spectrophotometrically, as described by Beers & Sizer (1952). The red cells of all species were diluted 1:500 with 0.05 molar phosphate buffer, pH 7.0, with the exception of the cells from the butterfly fish. The latter were diluted 1:2000 to overcome an apparent interfering effect on the catalase-peroxide reaction occurring at lower dilutions. The catalase activity is expressed as the velocity constant K x 10 -2. RESULTS Adding hydrogen peroxide to the red cell preparations produced the following reactions: the milkfish and acatalasemic mice, 0 - 1 + ; palani, 2 - 3 + ; naso, 3 + ; and butterfly fish and normal human, 4 + . Color changes in thawed red cell preparations were as follows: milkfish, dark brown-black; palani and naso, very dark red; and butterfly fish, reddish brown. All preparations were originally a moderately red color. Spectrophotometrically, neither the milkfish nor acatalasemic m i c e gave detectable activity. The remaining species gave highly variable results, with the palani and naso indicating low but close levels of activity, 1 and 2 respectively, and butterfly fish and normal humans the highest levels, 5 and 7 respectively. 331
332
A.C. SMITH DISCUSSION
Catalase activity in the fishes studied showed marked variation, consistent with the findings of Rabie et al. Further, the variations correlated with taxonomic relationships. This correlation was most apparent with respect to the similar activity readings, as well as the red cell color changes, of the palani and naso, both species being in the Family Acanthuridae (Lagler et al., 1963). The other fish species, all in different Families, gave significantly different readings and color changes. Most dramatic was the discovery of zero to minimal catalase activity in the red cells from the milkfish. As this species is a primitive teleostean, the initial appearance in evolution of the enzyme may have been in a later appearing, more advanced teleostean type. Fishes investigated to date in the latter category already demonstrate definite activity of this enzyme. Acatalasemic mice have been useful in biochemical genetic, etc. studies of this condition in humans. However, alternate species might also prove of value. Polypterus, a naturally acatalasemic animal as mentioned, is not readily available, at least not to most workers in the U.S.A. and probably not to many in other parts of the world. Milkfish, in contrast, is a more widely available animal. This species is a brackish water type and is commonly caught by fishermen in bays and a short distance offshore. Further, the milkfish is an important aquaculture species, being held in ponds for rearing purposes in many parts of the world. It, therefore, appears to be a potentially very useful and readily available model of a- or hypocatalasemia for research purposes. SUMMARY
AND CONCLUSIONS
1. Catalase activity was measured in the red cells of three modern and one primitive teleost--the milkfish, C. chanos.
2. The catalase activity and associated color changes of red cell preparations exposed to air correlated with the taxonomic relationships of the four fish species studied. 3. The modern teleosts investigated in this and the only other known study (Rabie et al., 1972) demonstrated definite enzyme activity, but the milkfish revealed virtually none. 4. Catalase, therefore, may have evolved with the higher teleosts. 5. The milkfish appears to provide an excellent and practical model of a- or hypo-catalasemia. The species is readily available in many parts of the world directly by catching from brackish sea water, or from aquaculture organizations rearing the animals. 6. Future studies on such animals as the milkfish may reveal how cells dispose of hydrogen peroxide in the virtual absence of catalase. Acknowledyements---The author wishes to thank the Oceanic Institute, Waimanalo, Hawaii, for providing the fish blood used in this study; Mr. Robert Matsuwaka and Ms. Ann Goishi, University of Hawaii, Honolulu, for excellent technical assistance in the laboratory; Dr. Douglas 'C. Vanm Associate Professor of Genetics, John A. Burns School of Medicine, University of Hawaii, for helpful comments on the manuscript; and the Department of Pathology, Saint Francis Hospital-John A. Burns School of Medicine, University of Hawaii, for the Pathology Research Fellowship that supported this study.
REFERENCES BEERS R. JR. • SIZER J. W. (1952) Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 195, 133-t40. LAGLER K. F., BARDACHJ. E. & MILLER R. (1963) Ichthyology, p. 132. John Wiley & Sons, New York, RABIE F., MAGID A. M. A., GUMA~, K. A. 8,z KARRARO. (1972) Evolution of catalase in fish. Comp. Biochem. Physiol. 43A, 1053-1055.
CATALASE IN FISH RED BLOOD CELLS A. C. SMIrd Department of Pathology, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822, U.S.A.
(Received 19 August 1975) Abstraet--Catalase activity was measured in the red blood cells from four different teleostean species: the milkfish, Chanos chanos; palani, Acanthurus dussumieri; naso, Naso lituratus; and butterfly fish, Chaetodon miliaris. 2. The enzyme activity and associated red cell color changes on exposure to air, correlated with the taxonomic relationships of the species studied. 3. Catalase was detected in all species except the most primitive--the milkfish--wh~, h revealed zero to minimal activity. These results are consistent with those of the only other known study (Rabie et al., 1972), on different species. 4. Catalase, therefore, may have evolved with the higher teleosts. 5. The milkfish is suggested as a possible model for a- or hypo-catalasemia in humans. INTRODUCTION The only report, to the author's knowledge, describing catalase investigations on the red blood cells of fishes is by Rabie et al. (1972). These authors worked with one of two living representatives of the primitive chondrosteans, Polypterus senegalus, and three modern teleosts (Tilapia nilotica, Clarias lazera, and Synodontis shall). Results indicated no catalase activity in the red cells of Polypterus, but definite, although variable, activity in the red cells of the other species. In Hawaiian waters, representatives of both primitive and modern teleostean fishes are available. Included in the former category is the milkfish, Chanos chanos. This species is intermediate in evolutionary position between the primitive and modern fishes of the study by Rabie et al. In the latter category are a great variety of reef fishes. These Hawaiian fishes could be a resource to extend studies of the taxonomic distribution of catalase, with its concomitant evolutionary implications, as begun by the authors mentioned. Further, the local availability of an a- or hypo-catalasemic fish would greatly facilitate medically oriented research underway on catalase in general. This work currently utilizes specially bred acatalasemic mice. MATERIALS A N D M E T H O D S
Fishes studied All fishes were collected in Hawaiian waters. Three individuals of each of the following species were used: milkfish, C. chanos; palani, Acanthurus dussumieri; naso, Naso lituratus; and butterfly fish, Chaetodon miliaris. Blood Sampling Blood samples were obtained by severing the caudal peduncle with a knife and collecting the blood from each species in a vial containing approx 0.2 ml of a super-saturated solution of the anticoagulant, EDTA*. The blood samples were centrifuged at approx 1171 g for five min at * Ethylenediaminetetracetic acid.
room temperature (25°C), and the supernatant plasma was discarded. The cells were then alternately washed with saline (0.85 g % NaC1) solution and centrifuged as before, 3-times. After the last centrifugation, the saline wash solution was discarded and the remaining blood cells were stored frozen until ready for testing.
Testing for Catalase (a) Subjective method. One drop of 3% hydrogen peroxide was added to one drop of the thawed blood (red) cell preparation on a glass slide. The reaction, in terms of amount of bubbling, was ranked 0 (no reaction) to 4 + (maximum bubbling, equal to that produced when normal human red cells are used). Red cells pooled from acatalasemic mice and normal humans served as controls. In addition, observations were made on the thawed red cells exposed to the air for a period of up to approx 2½ hr. The appearance of any color changes was noted. (b) Objective method. This procedure was carried out spectrophotometrically, as described by Beers & Sizer (1952). The red cells of all species were diluted 1:500 with 0.05 molar phosphate buffer, pH 7.0, with the exception of the cells from the butterfly fish. The latter were diluted 1:2000 to overcome an apparent interfering effect on the catalase-peroxide reaction occurring at lower dilutions. The catalase activity is expressed as the velocity constant K x 10 -2. RESULTS Adding hydrogen peroxide to the red cell preparations produced the following reactions: the milkfish and acatalasemic mice, 0 - 1 + ; palani, 2 - 3 + ; naso, 3 + ; and butterfly fish and normal human, 4 + . Color changes in thawed red cell preparations were as follows: milkfish, dark brown-black; palani and naso, very dark red; and butterfly fish, reddish brown. All preparations were originally a moderately red color. Spectrophotometrically, neither the milkfish nor acatalasemic m i c e gave detectable activity. The remaining species gave highly variable results, with the palani and naso indicating low but close levels of activity, 1 and 2 respectively, and butterfly fish and normal humans the highest levels, 5 and 7 respectively. 331
332
A.C. SMITH DISCUSSION
Catalase activity in the fishes studied showed marked variation, consistent with the findings of Rabie et al. Further, the variations correlated with taxonomic relationships. This correlation was most apparent with respect to the similar activity readings, as well as the red cell color changes, of the palani and naso, both species being in the Family Acanthuridae (Lagler et al., 1963). The other fish species, all in different Families, gave significantly different readings and color changes. Most dramatic was the discovery of zero to minimal catalase activity in the red cells from the milkfish. As this species is a primitive teleostean, the initial appearance in evolution of the enzyme may have been in a later appearing, more advanced teleostean type. Fishes investigated to date in the latter category already demonstrate definite activity of this enzyme. Acatalasemic mice have been useful in biochemical genetic, etc. studies of this condition in humans. However, alternate species might also prove of value. Polypterus, a naturally acatalasemic animal as mentioned, is not readily available, at least not to most workers in the U.S.A. and probably not to many in other parts of the world. Milkfish, in contrast, is a more widely available animal. This species is a brackish water type and is commonly caught by fishermen in bays and a short distance offshore. Further, the milkfish is an important aquaculture species, being held in ponds for rearing purposes in many parts of the world. It, therefore, appears to be a potentially very useful and readily available model of a- or hypocatalasemia for research purposes. SUMMARY
AND CONCLUSIONS
1. Catalase activity was measured in the red cells of three modern and one primitive teleost--the milkfish, C. chanos.
2. The catalase activity and associated color changes of red cell preparations exposed to air correlated with the taxonomic relationships of the four fish species studied. 3. The modern teleosts investigated in this and the only other known study (Rabie et al., 1972) demonstrated definite enzyme activity, but the milkfish revealed virtually none. 4. Catalase, therefore, may have evolved with the higher teleosts. 5. The milkfish appears to provide an excellent and practical model of a- or hypo-catalasemia. The species is readily available in many parts of the world directly by catching from brackish sea water, or from aquaculture organizations rearing the animals. 6. Future studies on such animals as the milkfish may reveal how cells dispose of hydrogen peroxide in the virtual absence of catalase. Acknowledyements---The author wishes to thank the Oceanic Institute, Waimanalo, Hawaii, for providing the fish blood used in this study; Mr. Robert Matsuwaka and Ms. Ann Goishi, University of Hawaii, Honolulu, for excellent technical assistance in the laboratory; Dr. Douglas 'C. Vanm Associate Professor of Genetics, John A. Burns School of Medicine, University of Hawaii, for helpful comments on the manuscript; and the Department of Pathology, Saint Francis Hospital-John A. Burns School of Medicine, University of Hawaii, for the Pathology Research Fellowship that supported this study.
REFERENCES BEERS R. JR. • SIZER J. W. (1952) Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 195, 133-t40. LAGLER K. F., BARDACHJ. E. & MILLER R. (1963) Ichthyology, p. 132. John Wiley & Sons, New York, RABIE F., MAGID A. M. A., GUMA~, K. A. 8,z KARRARO. (1972) Evolution of catalase in fish. Comp. Biochem. Physiol. 43A, 1053-1055.
