Breed differences in sheep erythrocyte carbonic anhydrase activity

Comp. Biochem. Physiol., 1970, Vol. 35, pp. 639 to 646, Pergamon Press. Printed in Great Britain

BREED D I F F E R E N C E S IN SHEEP ERYTHROCYTE CARBONIC ANHYDRASE A C T I V I T Y N. S. AGAR, J. R O B E R T S and J. V. EVANS Department of Physiology, University of New England, Armidale, N.S.W. 2351 (Received 17 January 1970)

Abstract--1. Four breeds of sheep (Merino, Corriedale, Dorset Horn and Border Leicester) have been examined for erythrocyte carbonic anhydrase activity. 2. Significant between-breed differences have been found. 3. A relationship has been demonstrated between carbonic anhydrase activity and age, up to 10 weeks. 4. Haemolytic anaemia has been shown to reduce carbonic anhydrase activity per/zl red blood cells by almost 50 per cent. 5. Carbonic anhydrase activity was examined in relation to haemoglobin type and potassium type. No significant relationships were found. 6. Only one fraction showing carbonic anhydrase activity was demonstrated electrophoretically. INTRODUCTION SINCE TUE discovery of carbonic anhydrase in mammalian erythrocytes by Meldrum & Roughton (1932a) the distribution, chemistry, physiology and pharmacology of the enzyme have been investigated extensively (Meldrum & Roughton, 1932b, 1934; Maren, 1967). However, there appears to be only one report on the level of carbonic anhydrase activity in sheep erythrocytes and only two on the electrophoretic separation of the enzyme in sheep haemolysates in the literature. Larimer & Schmidt-Nielsen (1960) compared the carbonic anhydrase activity of the erythrocytes of various mammalian species and reported that, in general, red cells of small animals had a higher enzyme activity than those of larger animals. They noted, however, that sheep were an exception to this general rule and that the range of carbonic anhydrase activity in sheep was greater than in other animals. Tucker et al. (1967) demonstrated two isozymes electrophoretically in sheep erythrocytes and called them "carbonic anhydrase fast moving" (CAF) and "carbonic anhydrase slow moving" (CAS). Only one sheep out of 696 had the CAF isozyme. Tucker (1967) reported that only the CAS isozyme was present in forty-two sheep of five rare breeds. Hilkovitz (1957) demonstrated that acetazolamide, a powerful and specific inhibitor for carbonic anhydrase, reduces sickling of human erythrocytes both in vivo and in vitro. His in vivo studies have been questioned by Henderson et al. (1959), Holder & Hayes (1965) and Basu & Woodruff (1966). However, based on 639

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his unpublished data, M a r e n (1967) remarked, "it is not possible at present to draw up any reasonable scheme that would explain a reduction of sickling through inhibition of carbonic anhydrase. Possibly there is interaction between acetazolamide and haemoglobin, which should be explored." Until H o l m a n & D e w (1964) described sickling in 1 - 3 - m o n t h - o l d goats, this p h e n o m e n o n had been observed only in m a n and deer. Recently Evans (1968) demonstrated sickling in sheep erythrocytes. H e found that thirty-eight out of thirty-nine Soay sheep, all twelve Swaledale lambs and three out of nine adult Swaledale sheep showed this characteristic in their erythrocytes. N o n e of the twelve Clun Forest, however, had siekled red cells. T w o simply inherited biochemical characteristics of sheep, blood potassium types (Evans, 1954) and haemoglobin types (Harris & Warren, 1955; Evans et al., 1956) are well known. Because of the lack of information relating to the level of carbonic anhydrase in the erythrocytes of sheep, the great variation in the enzyme activity observed by L a r i m e r & Schmidt-Nielsen (1960) and the possibility of a relationship between carbonic anhydrase levels and the haemoglobin type and/or blood potassium type, it was decided to investigate these parameters in sheep of different breeds and ages. A brief communication has already been made (Agar et al., 1969). MATERIALS AND M E T H O D S Adult ewes (Ovis aries) of four different breeds, Border Leicester, Corriedale, Dorset Horn and Merino, and lambs of the Dorset Horn and Merino breeds were used. The animals were made available by the C.S.I.R.O., Division of Animal Physiology, Pastoral Laboratory, Armidale, N.S.W. All the ewes had been mated and were maintained under similar nutritional and environmental conditions. Blood was obtained by jugular venepuncture and collected into a heparinized universal container. One ml of blood was diluted to 100 ml with ice-cold deionized water. Carbonic anhydrase activity was measured at 0°C according to a modification of the method of Maren (1960). The blank consisted of 0"4 ml indicator buffer, 0"3 ml deionized water and 0"1 ml carbonate-bicarbonate buffer. For the blood samples, the 0'3 ml of water was replaced by 0-27 ml water plus 0"03 ml of 1 : 100 diluted blood. Thus the final volume was 0"8 ml in each case. On addition of the 0"1 ml carbonate-bicarbonate buffer a stopwatch was started, and the time taken for the mixture to become acid (indicated by a red/yellow colour change of the indicator) was noted. At the end of each run the contents of the vessel were emptied by suction and the vessel rinsed three times with deionized water. All the reagents including the blood samples w e r e k e p t in ice cold water. A blank reading was obtained by taking the average of five to twelve readings each day, while the experimental reading was an average of three to five readings. Most of the estimations were done on the day of blood collection although it was noted that there was no change in the enzyme activity when diluted blood was stored at 4-5°C for 2-3 weeks. Enzyme activity was calculated according to the following formula : 1 unit of enzyme activity - To - T T ' where T is the time in seconds for the mixture to become acid in the presence of the enzyme and To is the time in its absence. The enzyme activity was then expressed as units//xl red blood cells (E.U//zl RBC).

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Packed cell volume was determined by the microhaematocrit method. Na + and K + concentrations in whole blood and plasma were determined by flame photometry using a Technicon Autoanalyser. Erythrocyte values for Na + and K + were calculated by difference. Hb type was determined by paper electrophoresis using Tris-borate buffer, pH 9-2. Starch-gel electrophoresis was used to locate the bands showing carbonic anhydrase activity. The discontinuous buffer system adopted was that of Ferguson & Wallace (1963). The staining and inhibitor tests used were those described by Tucker et al. (1967). RESULTS I t is evident f r o m T a b l e 1 that the m e a n level of carbonic anhydrase activity in the erythrocytes was different in the breeds studied. T h e Merino breed had the highest m e a n value and the Corriedales had the lowest. Border Leicester and Dorset H o r n sheep had intermediate values. I t is also interesting to note that sheep of the Merino and Corriedale breeds had the highest range of activity while this range was comparatively small in the Border Leicester and Dorset H o r n breeds. An analysis of variance showed a significant between-breed difference in carbonic anhydrase activity. W h e n breed means were c o m p a r e d using D u n c a n ' s multiple range test (Duncan, 1955) there were significant differences at the 1 per cent level between breeds as shown in T a b l e 1. T A B L E 1 - - B R E E D DIFFERENCES I N MEAN CARBONIC ANHYDRASE LEVELS I N SHEEP ERYTHROCYTES

(enzyme units//~l red blood cells) Breed Corriedale Dorset Horn Border Leicester Merino

No. of animals

Range

Mean ( + S.E.M.)

25 25 25 25

3"46-11"61 4"15-9"11 4"92-10"14 4-70-15"09

6"42 ( + 0.41)~ 7"35 ( _+0"29)J I 8.21 ( +_0'30) t/ 9.31 ( +_0"57) /

Braces connect means which are not significantly different. T a b l e 2 shows the erythrocyte carbonic anhydrase levels of sheep of different haemoglobin types. T h e s e data did not indicate an association between haemoglobin type and carbonic anhydrase activity, although the small n u m b e r of H b A animals did not allow a good comparison to be made. T A B L E 2 - - C A R B O N I C ANHYDRASE LEVELS I N THE ERYTHROCYTES I N RELATION TO HAEMOGLOBIN TYPE OF SHEEP

Mean carbonic anhydrase level (E.U//~I RBC) Breed Merino Border Leicester Dorset Horn Corriedale

Hb A

Hb AB

Hb B

9"19 (2) -6"45 (2) --

9"68 (14) 8"24 (5) 7-76 (8) 6"30 (9)

8-76 (9) 8"22 (20) 7"26 (15) 6"49 (16)

No. of animals in parenthesis.

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In the present study, only the Dorset Horn flock contained H K animals (Evans, 1954). In a previous experiment the carbonic anhydrase levels in H K and L K animals of the Merino breed had been determined. These data have been included in this work. The levels of carbonic anhydrase activity of L K and H K sheep are presented in Table 3. Akhough in both the Dorset Horn and the Merino breed, the values for H K animals are higher than those for L K animals, the differences are very small. T A B L E 3 - - C A R B O N I C ANHYDRASE LEVELS IN THE ERYTHROCYTES IN RELATION TO BLOOD POTASSIUM TYPE OF SHEEP

Mean carbonic anhydrase level (E.U//zl RBC) Breed Merino Dorset Horn

HK

LK

8.73 (24) 7'69 (7)

8"59 (119) 7-22 (18)

No. of animals in parenthesis. The mean carbonic anhydrase level in 1-4-week-old Dorset Horn lambs was 2.96 E.U//xl RBC (Table 4). This is approximately one-third of the adult level found in this experiment (Table 1). Lambs between 6-10 weeks of age gave a mean level of 5.75 E.U. which is about two-thirds of the adult level. Merino lambs of 1-4 weeks of age gave a mean value of 2.72 E.U. which corresponds very well with the results obtained with the Dorset Horn lambs. T A B L E 4 - - - C A R B O N I C ANHYDRASE LEVELS IN THE ERYTHROCYTES OF LAMBS

Carbonic anhydrase level (E.U//zl RBC) Breed Dorset Horn Dorset Horn Merino

No. 8 11 12

Age in weeks

Mean

Range

S.E.M.

6-10 14 14

5"75 2"96 2' 72

4'27-7"07 1"84--5'07 1.494.19

0"38 0"28 0"26

When the erythrocytes of four normal adult Merinos (two Hb A and two Hb B) were monitored for carbonic anhydrase activity over a period of 7 weeks, the levels remained relatively constant. Two of these sheep (one Hb A and the other Hb B) were then made anaemic by repeated bleeding over a period of ten days. The carbonic anhydrase levels (activity//xl RBC's) at the end of this period were about 50 per cent of their pre-anaemic values (Table 5). Hb C was detected in the Hb A animal and not in the Hb B animal. Starch gel electrophoresis revealed the presence of only one zone having carbonic anhydrase activity. This zone was present behind the haemoglobin zone,

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SHEEP ERYTHROCYTE CARBONIC ANHYDRASE ACTIVITY

as shown in Fig. 1. No carbonic anhydrase polymorphism was observed in the present experiment. T A B L E 5 - - C H A N G E S I N CARBONIC ANHYDRASE ACTIVITY D U R I N G ANAEMIA

Sheep Control A B Exp. A B

Pre-anaemic values mean of 5 readings ( + S.E.M.)

Anaemia production

Post-treatment values Day 1

Day 3

Day 5

Day 7

3"26 + 0"18 8"26 + 0"15 2"78 + 0"10 10"04 + 0'18

+ +

2'80 7"70 1"50 6"20

3"50 8'00 1"40 6"50

3'90 8.00 1"70 6"50

3-20 8-50 1"75 7'00

DISCUSSION The four breeds of sheep examined had different mean levels of carbonic anhydrase activity in their erythrocytes. As nutritional and environmental factors were standardized as far as possible, these factors would not appear to be responsible for the observed differences. Genetic factors, or an interaction between genetic and environmental factors could be involved. Larimer & Schmidt-Nielsen's (1960) report appears to be the only reference in the literature to the carbonic anhydrase activity of sheep erythrocytes. Their results cannot be compared directly with those given in this paper because different buffer systems were used in the two experiments. However, Maren (1967) suggested that 1000 enzyme units of the bicarbonate buffer system (used in the present investigation) are equivalent to 3000 E.U. of the barbital buffer system (used by Larimer & Schmidt-Nielsen). Using this comparison, their mean value for five sheep of unspecified breed is 7.05 E.U/~I RBC with a range of 4.82 to 8.83 E.U/#I RBC. This value is within the range of values for the four breeds studied in this experiment. There are two main haemoglobin types in sheep--Hb A and Hb B. These haemoglobins differ in their oxygen dissociation curves and solubilities (van der Helm et al., 1957; Huisman et al., 1958; Dawson & Evans, 1962) and in their electrophoretic and chromatographic behaviour, amino acid composition and sequence (Harris & Warren, 1955; Evans et al., 1956; Huisman, 1966). However, the results presented in Table 2 do not suggest a relationship between sheep haemoglobin types and carbonic anhydrase activity. The role of K +, Hb and carbonic anhydrase in the uptake and delivery of CO 2 and 02 is well known. In sheep K + concentrations in the erythrocytes show a bimodality being either 10-15 m-equiv/1. RBC (called LK) or 60-90 m-equiv/1. RBC (called HK) (Evans, 1954). This large difference in K + concentrations in the erythrocytes of sheep could conceivably be associated with differences in carbonic anhydrase activity. This hypothesis is supported by the observations of Korman & Kera (1961) who reported that when intact human red cells were incubated with K + at 37°C for 20 min in vitro, there was an increase in the carbonic anhydrase

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activity. The absence of any association between the potassium type and the carbonic anhydrase activity in the present experiment (Table 3) indicates that large differences in the K + concentration in the erythrocytes have no effect on the activity of the enzyme. As early as 1934 Meldrum & Roughton found that goat foetuses were low in carbonic anhydrase and stated, "In the very young foetuses, there is extraordinarily little enzyme and the amount does not begin to rise appreciably until very near the end of term". Similar observations have been reported in man (Stevenson, 1943; Berfenstam, 1952; Maren, 1967), rhesus monkey (Fisher, 1961) and mice (Clark, 1951). It is possible that these low foetal levels of carbonic anhydrase are associated with the presence of foetal haemoglobin. In the camel, however, lower foetal levels of carbonic anhydrase do not appear to be associated with the presence of Hb F (Riegel et al., 1967). From the results presented in Table 4, it appears that in sheep, carbonic anhydrase activity rises quite rapidly in the first few months of life. During this period the levels of Hb F are decreasing. Starch-gel electrophoresis showed that Hb F was present in the blood of 1-4-week-old lambs but not in the blood of those 6-10 weeks old. In all mammals and birds, foetal blood has a higher O s affinity than maternal blood. There is no doubt that this is advantageous to the foetus. On the other hand, there are theoretical reasons why a low blood level of carbonic anhydrase as found in foetal and early post-natal life might interfere with the oxygenation of the blood. Oxygenation of haemoglobin is physiologically speeded up when the blood gives up carbon dioxide in the lungs. Also the haemoglobin O s dissociation curve is shifted to the left proportional to the lowering of the CO s tension. It is possible that a low blood level of carbonic anhydrase delays the lowering of the CO s tension in the lung capillaries, resulting in a slow and incomplete oxygenation of haemoglobin. Maren (1967) stated, "The interesting question is raised of the relationship between the foetal pattern for haemoglobin and carbonic anhydrase, although the synthesis of these two proteins is under separate genetic control". Vallee et al. (1949) reported that in various forms of anaemia in man the erythrocyte carbonic anhydrase activity did not change. In pernicious anaemia, however, they found that the enzyme activity per red cell doubled so that whole blood activity was about normal. Studies on the carbonic anhydrase levels in anaemic blood do not appear to have been made in other animals. In the present experiment, the erythrocyte carbonic anhydrase activity in the two anaemic sheep was found to be significantly lower than their pre-anaemic values. These observations have since been confirmed (Perry & Agar, unpublished data). Moreover, it also appears that Hb C which is produced in anaemic Hb A or Hb AB sheep and not in Hb B sheep (Blunt & Evans, 1963 ; van Vliet and Huisman, 1964; Agar & Evans, 1969) is not associated with carbonic anhydrase response since the proportional reduction of carbonic anhydrase values was similar in sheep of Hb A and Hb B types. The carbonic anhydrase activity was present in a single protein band migrating just behind the haemoglobin (Fig. 1). Its mobility was similar in sheep of different

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645

haemoglobin types. Similar mobilities have been reported in man and various primates (Haut et al., 1962; Tashian & Shaw, 1962; Tashian, 1965, 1968). T u c k e r et al. (1967), on the other hand, demonstrated two carbonic anhydrase isozymes of sheep erythrocytes, both of them in front of the haemoglobin bands. T h e p H of their buffers may have resulted in the greater mobility of the enzyme bands which they found compared to the one found in the present experiment. Acknowledgements--The authors have pleasure in acknowledging the skilled and willing technical assistance of Mr. J. Sheedy, the advice given by Dr. M. H. Blunt in the development of the electrophoretic techniques used in these experiments and the C.S.I.R.O., Division of Animal Physiology, for allowing them access to their flocks. This work was supported by grants from the Australian Research Grants Committee, the Australian Meat Research Committee and the University of New England.

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Key Word Index--Carbonic anhydrase in sheep; RBC; age and carbonic anhydrase activity; species differences in RBC carbonic anhydrase.