Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 83 to 87. Per#amon Press. Printed in Great Britain

EQUILIBRIA AND LIGAND BINDING KINETICS OF HEMOGLOBINS FROM THE SHARKS, P R I O N A C E G L A U C A AND CAR C H A R H I N US M I L B E R TI* RUSSELL R. PENNELLY, ROBERT W. NOBLE,t AND AUSTEN RIGGS From the Departments of Medicine and Biochemistry, SUNY at Buffalo, the Veterans Administration Hospital, Buffalo, NY 14215, and the Department of Zoology, University of Texas at Austin, Austin, TX 78712, U.S.A.

(Received 11 October 1974) Abstract--1. The functional behavior of the hemoglobins from two sharks (Prionace glauca and Carcharhinus milberti) obtained off-shore from Kealakakua Bay, Hawaii, has been examined: 2. Neither hemoglobin shows an apparent Root effect. The binding properties in general are very similar to those of mammalian hemoglobins. 3. The oxygen equilibria of the maternal and fetal hemoglobins from C. milberti have been compared. The results show that fetal hemoglobin has a slightly higher oxygen affinity in the absence of endogenous phosphate, but the affinities of fetal and maternal hemoglobins appear identical in 0.8 mM ATP.

INTRODUCTION

kinetics of the reactions of these hemoglobins with oxygen and carbon monoxide. In addition a small quantity of fetal sand bar shark hemoglobin was obtained for study. A comparison of the oxygen affinity of this fetal hemoglobin to that of the adult of the species is also reported.

THE EQUILIBRIUMand kinetic properties of the ligand binding reactions of the hemoglobins isolated from a number of teleost fish have been reported (see for example Brunori et al., 1973, Tan et al., 1973, Gillen et al., 1973, and Lau et al., 1975). These hemoglobins are found to have widely varying functional properties which have offered new insights into the range of behaviors obtainable from tetrameric hemoglobins. Certain of these properties, such as the Root effect, can be related directly to physiological needs unique to fish, such as swim bladder filling. The origins of others are less apparent. Because of the numerous informative properties discovered in studying the hemoglobins of teleost fish, it was of obvious interest to examine the properties of the hemoglobins of some elasmobranchs. These fish lack swim bladders, but it seemed possible that they would have physiological requirements which would place unique constraints on the functional properties of their hemoglobins. This paper is a report on investigations which have been made on two shark hemoglobins. Both blue shark (Prionace glauca) and sand bar shark (Carcharhinus milberti) hemoglobins were obtained during the expedition. We have measured the equilibria and

MATERIALS A N D

METHODS

Isolation Blood from adult sharks was collected either by severing the tail and collecting the outflow from the spinal artery or by decapitation and collecting the blood from the carotid arteries. The fish were caught off Kealakakua Bay, Hawaii, during the Kona Coast Alpha Helix Expedition (1973). Cells from fetal Carcharhinus milberti were obtained by severing the cord between the yolk sac and the fetus and by direct dissection. Blood was collected in Alsever's solution (Kolmer, 1949). Cells from Prionace glauca were washed three times with Alsever's solution, then either frozen in liquid nitrogen as packed cells or lysed by addition of 1 mM Tris, pH 8 followed by standing at 4°C for 30 min. Cells of C. milberti were very fragile and were washed only once before storage or lysis. Stroma was removed from the hemolysates by addition of NaCl to a final concentration of 0.1 M and centrifugation at 25,000 x 9' for 20 min. Hemolysates prepared in this manner were then stripped of endogenous phosphates by passage through a 2.5 x 100 cm G-100 Sephadex column previously equilibrated with a l mM Tris, 0.1 M NaC1, pH 8 buffer at a flow rate such that the elution time was 6--7 hr. Previous control experiment~ showed that this procedure was sufficient to remove all endogenous phosphate.

* This work was supported by the National Science Foundation under grants NSF-GB36349, GD 34462-1 and GB 39268 to the Scripps Institute of Oceanography for operation of the Alpha Helix Research Program. Also supported by research grant HL 12524 from the Heart and Lung Institute of the National Institute of Health (to R.W.N.), funds from Veterans Administration Research Project 6098-01 (to R.W.N.), NSF grant GB 27937X (to A.R.) and a grant from the Universfty of Texas Research Institute (to A.R.). t This work was carried out during the tenure of an American Heart Association Established Investigatorship.

Gel electrophoresis Vertical polyacrylamide gel electrophoresis was done at 4-6°C at 18 V/cm with a continuous buffer system of TrisEDTA-borate buffer, pH 8.9 as described by Raymond (1962). 83

R, R. PENNELLY,R. W. NOBLE AND A. RlGGS

84

90

Ligand bindin9 equilibria Oxygen equilibria were performed at 20°C with a modification (Nagel et al., 1965) of the Allen, Guthe & Wyman (1950) method. Solutions were approximately 0.25 mM in heme equivalents. The hemoglobin was deoxygenated and titrated with a previously prepared solution of dithionite in deoxygenated water. This reduced any methemoglobin to the ferrous derivative and prevented its further formation.

5O 50

2O

~1o

Stopped-Flow kinetic measurements Oxygen dissociation was measured using a Gibson & Milnes (1964) stopped-flow apparatus. One solution was prepared by placing hemoglobin in 1 mM Tris buffer also containing NaC1 and, when used, IHP (inositol hexaphosphate). This solution was then mixed in the stopped-flow with dithionite dissolved in 0.1 M buffer of the desired final pH. After mixing, the concentration of NaC1 was 0.1 M. For P. 91auca the IHP concentration after mixing was 1 mM and for C. milberti 0.25 mM. The reactions were followed at 535, 578 and 560 nm and both 2 and 20°C for blue shark hemoglobin and at 435 nm and 20°C for sand bar hemoglobin. The final hemoglobin concentration in either case was between 25 and 60 #M. Carbon monoxide combination experiments were also carried out with the stopped-flow apparatus at 20°C. A CO saturated water solution was diluted into deoxygenated buffers and this solution was mixed with deoxygenated hemoglobin again buffered in 1 mM Tris with NaC1 and IHP as in the 02" dissociation experiments. The reaction was observed at 420 nm. The final hemoglobin concentration was 1-2 pM and the CO concentration was 50 pM. The results shown represent the rate through 50% of the reaction.

Flash photolysis kinetic measurements The CO recombination rates due to both full and partial flash photolysis were measured with an apparatus described previously (Lau et al., 1975). The reaction solution was prepared by injecting anaerobically an appropriate amount of CO saturated water into deoxygenated buffer. This was followed by anaerobic injection of hemoglobin and then by the addition of dithionite to absorb residual oxygen and to maintain the deoxygenated status of the reaction. This solution was mixed well and then flushed carefully into a 1 cm, water jacketed, sample cuvette and positioned in the flash apparatus. The instrument operation is described elsewhere in this journal (Noble et al., 1975).

Buffers

5 D

3 2

Fig. 1. The pH dependence of the oxygen affinity of P. bin (O O) and for hemoglobin in the presence of 1 mM IHP (~ 0). P1/2(02), the oxygen partial pressure required for half saturation, is plotted on a logarithmic scale as a function of pH. logarithmic scale vs pH. Data for stripped hemoglobin shows a 6-fold affinity range while the range for stripped hemoglobin with 1 m M I H P is a b o u t 16fold. The instability of the protein prohibited measurements at lower pH. The n-values computed from Hill plots of this data ranged from 1.1 to 1.9 a n d show a gradual decrease with increasing pH. The values obtained in the absence of organic phosphates are generally slightly higher t h a n those in the presence of IHP. The Hill plots were linear and were fitted by a least squares procedure in order to determine n a n d Pin(02). 0 2 affinity of Carcharhinus milberti hemoglobin Oxygen equilibria experiments were made on C.

milberti hemoglobin also as a function of p H a n d the results are shown in Fig. 2. The data for b o t h stripped hemoglobin a n d stripped hemoglobin with 0'25 m M I H P is shown plotted on the same logarithmic scale as the blue shark data. The affinity range for the stripped material is a b o u t 3-fold. While the total

30 20

\

IC

o7 ffJ"5 3

f

o

o->-.o o

*

2

7'o

RESULTS

ence a n d absence of organic phosphate. The results are given in Fig. 1 where P l n ( O 2 ) is plotted o n a

9.0 '

91auca hemoglobin. Data points are for stripped hemoglo-

8.o

pH

02 affinity of Prionace glauca hemoolobin Oxygen equilibria experiments were performed on

8.0 '

7.0

pH

For stripped hemoglobin 0.05 M, Tris (pH 8-9), Bi~Tris (pH 6-8), and cacodylate (pH 5-6) buffers were used. Phosphate (pH 6-8), citrate (pH 5-6), and borate (pH 8-9) buffers of the same concentration were used for hemolysate experiments or those in which IHP was present. In all experiments other than oxygen dissociation, NaCI was added to the buffer so that the final concentration would be 0.1 M. In the presence of IHP this concentration of NaCI had little effect. IHP was also added to the buffer such that its final concentration was 1 mM in the case of P. olauca and 0-25 mM for C. milberti. In those experiments requiring deoxygenation, the buffer was equilibrated with water saturated oxygen free hydrogen.

P. olauca hemoglobin as a function of p H in the pres-

6D '

I

Fig. 2. The effect of pH on the oxygen affinity of C. milberti hemoglobin. Data is presented for stripped hemoglobin (O

O) and for stripped hemoglobin in the presence of 0.25 mM IHP (,O).

Hemoglobins from sharks

85

40

1.2 .8

--

.I

?

.4

30

\

o

1

o O

-.4

20 o

15 o

6.0

-8

7.0

8.0

0

pH

-I.2 I

I

I

I

I

I

0.2 g.6.8 tO Log P(O2) Fig. 3. Comparison of the oxygen affinities of adult and fetal hemoglobins of C. milberti. The data are presented as Hill plots for the binding of oxygen to the fetal (O O) and adult ([] [3) hemoglobins in the absence of organic phosphates and to the fetal (,ID) and adult (1 l) hemoglobins in the presence of 0.8 mM ATP.

range for the stripped hemoglobin with IHP cannot be estimated from this data, a 10-fold range is seen. The n-values for C. milberti range from 2'0 to 3"0 and show a similar decrease with pH as did the P. glauca values. The lower values for the blue shark may indicate the presence of hemoglobin fractions with dissimilar functional properties, or ct chain-fl chain heterogeneity.

Comparison of the oxygen affinities of fetal and adult Carcharhinus milberti hemoglobin The oxygen affinities of fetal and adult C. milberti hemoglobins, stripped and in the presence of 0'8 mM ATP at pH 6"9 were measured and compared. The Hill plots are shown in Fig. 3. All slopes are nearly equivalent with n-values near 2'3. When stripped of organic phosphates, fetal hemoglobin has a higher oxygen affinity compared to that of adult hemoglobin. The addition of ATP lowers the affinities of both hemoglobins to the same level.

140 !

0o jo _~

Fig. 5. The dependence of the 02 dissociation rate, k, of C. milberti hemoglobin on pH. These data are plotted on the same logarithmic scale as the P. #lauca results for purposes of comparison. Again, the rates for both stripped hemoglobin (O O) and stripped hemoglobin with 0-25 mM IHP (,0) are shown.

Rates of globin

0 2

dissociation from Prionace glauca hemo-

The pH dependence of the 02 dissociation reaction, k, is shown in Fig. 4. There is an approximate 2-fold range of rates between pH 8.5 and pH 6"0. The maximum rate is 105 sec-1 at pH 6 and a slight acid Bohr effect is seen at lower pHs. The addition of 1 mM IHP increases the rate, as shown by the data between pH 6.5 and 7.5, by a constant 35~o.

Rate of 02 dissociation of Carcharhinus milberti hemoglobin The full pH profiles of the rates of 02 dissociation from C. milberti hemoglobin, both stripped and stripped with 0.25 mM IHP, are shown in Fig. 5. The results are plotted on the same scale as the data for P. glauca. The 02 dissociation rates for C. milberti hemoglobin exhibit the same variation with pH as the blue shark hemoglobin with the maximum rate again seen near pH 6. The higher affinity of C. milberti observed in the equilibrium measurements is reflected in the slower dissociation rates, since the maximum rate at pH 6 is 34 sec-t. IHP raises the rates at all pH's. From pH 5 to 7 the increase is about

4

"\

o

\

A

.,/

o

,o.,

to

to

.~ 70 'O 4

50

o~o__o

40

ob

I

pH

7.0

d.0

Fig. 4. The dependence of the 02 dissociation rate of P.

I

~

I

,

i

L

5.0

6.0

7.0 pH

8.0

9.0

olauca hemoglobin on pH, k, the rate constant for this reaction is plotted on a logarithmic scale as a function of pH. Results for stripped hemoglobin (O O) and stripped hemoglobin in the presence of 1 mM IHP (~ 0) are shown.

Fig. 6. The effect of pH on the rate of carbon monoxide combination to P. glauca hemoglobin both stripped of organic phosphates (O O) and in the presence of 1 mM IHP.

R.R. PENNELLY,R. W. NOBLEAND A. RIGGS

86 10

---5

h X

2

5.O

6.O

7.O pH

80

9.0

Fig. 7. The pH and phosphate dependence of the CO combination rate for C. milberti hemoglobin. The logarithmic scale is the same as used for the P. 9lauca l' data. Stripped hemoglobin (O ©) and stripped with 0.25 mM IHP (," , ) results are shown. 40~o, while at higher pH values a small increase is observed. Again for the sand bar shark there are data suggesting an acid Bohr effect. CO Combination kinetics measured by stopped-flow The pH dependence of the CO combination rate constant, l', for P. 9lauca is given in Fig. 6 and for C. milberti in Fig. 7. Both are plotted on the same scale coordinates. The values of these rates for both shark hemoglobins in both the presence and absence of IHP are nearly identical below pH 8. The l' data for the sand bar shark above pH 8-5 is extremely heterogeneous with a very rapid initial phase. However, even the slower phase of the reaction has a rate faster than that found for P..qlauca. The effect of IHP is to lower the combination rate at all pHs. The apparent minimum is shifted toward higher pH by about 0'4 units by the addition of IHP. The data are consistent with an increased affinity at low pH for both shark hemoglobins.

Flash photolysis measurements The rates of CO combination to both fully and partially liganded hemoglobin were measured over the entire pH range for C. milberti and at pH 6"5 for P. 91auca. Stripped hemoglobins in both the presence and absence of IHP were studied. Partial photolysis was done in an effort to get an estimate of the cooperativity of ligand binding. The rates of CO recombination after partial photolysis of stripped C. milberti hemoglobin with and without IHP are 5- to 10-fold faster than those measured after full flash in the pH range from 5 to 9. The blue shark hemoglobin at pH 6-5 shows similar results.

DISCUSSION

The hemoglobins of the elasmobranchs which we have studied do not exhibit unusual or unexpected functional properties. Both acid and alkaline Bohr

effects are seen and the kinetic effects of their changes in ligand affinity are similar to those found for mammalian hemoglobins. As might have been predicted from the absence of swim bladders in these animals, these hemoglobins do not display Root effects. Furthermore, although teleost hemoglobins often exhibit a loss of cooperative ligand binding at the extremes of ligand affinity, particularly the low affinity extremes, ligand binding to these shark hemoglobins was cooperative under all conditions examined. In comparing the hemoglobin of the blue shark with that of the sand bar shark many similarities are seen. The magnitude of the pH dependencies of the different equilibrium and kinetic constants measured are generally similar for these two hemoglobins. The pH dependences of P~/2(02) and k are slightly greater for P. glauca than for C. milberti, but this is a rather small difference. In spite of similar pH dependences, the hemoglobin of the blue shark consistently displays oxygen affinities that are approximately 2-fold lower than those of the hemoglobin of the sand bar shark. The differences in oxygen affinity observed, both between species and as a result of variations of pH and organic phosphate, are reflected in variations in the rate of oxygen dissociation, k. Thus, the rates of oxygen dissociation obtained from the hemoglobin of P. glauca are consistently faster than those for the hemoglobin of C. milberti. In contrast, the rates of CO combination for the two hemoglobins are very similar. They do differ above pH 8, but under these conditions the hemoglobin of C. milberti is kinetically very heterogeneous and it may be that dissociation of the tetramer occurs. No attempt was made to purify the hemoglobins of these sharks. If fractions of widely differing functional properties are present, this could complicate the interpretation of the results. This does not appear to be the case. The hemoglobin of P. glauca appeared as a single band on gel electrophoresis while that of C. milberti contains more than one component. However, the high values of n obtained with the latter hemolysate argue against large functional differences between these components. In mammals, 02 transport between parent and fetus is facilitated by the fetal blood having a higher affinity for oxygen than the adult blood. C. milberti is a live bearer and although sharks lack placental structures, oxygen transport must be accomplished between the maternal and fetal circulatory systems. We find that the fetal hemoglobin of C. milberti has a slightly higher 02 affinity than that of the adult in the absence of organic phosphates. Addition of ATP decreases both affinities and makes them similar or identical. We cannot say whether or not in vivo an Oz affinity difference is maintained, but by the appropriate regulation of organic phosphate levels, such a difference is clearly possible. Manwell (1958a, b; 1963) has reported similar differences between fetal and adult hemoglobins for two other elasmobranchs, the spiny dogfish (Squalus suckleyi) and the barn door skate (Raja binoculata). However, his measurements were made only in 0.1 M phosphate buffer plus any endogenous phosphate which may have been present.

Acknowledgement--We wish to thank Roger J. Wilson for technical assistance in making many of the equilibrium measurements.

Hemoglobins from sharks

87

MANWELLC. (1958a) A "Fetal-Maternal Shift" in the Ovoviviparous Spiny Dogfish, Squalus Suckleyi (Girard). ALLEN D. W., GUTn~ K. F. & WYMANJ. JR. (1950) Further Physiol. Zool. 31, 93-100. Studies on the Oxygen Equilibrium of Hemoglobin. J. MANWELL C. (1958b) Ontogeny of Hemoglobin in the Biol. Chem. 187, 393-410. Skate, Raja binoculata. Science, N.Y. 128, 419-420. BRUNORI M., BONAVENTURAJ., BONAVENTURAC., GIAR- MANWELL C. (1963) Fetal and Adult Hemoglobins of the DINA B., BOSSAF. & ANTONINIE. (1973) Hemoglobins Spiny Dogfish Squalus suckleyi. Archs Biochem. Biophys. from Trout: Structural and Functional Properties. 101, 504-511. Molec. Cell. Biochem. 1, 189-196. NAGEL R. L., WITTENBERGJ. B. & RANNEYH, M. (1965) GIBSON Q. H. & MILNES L. (1964) Apparatus for Rapid Oxygen Equilibria of the Hemoglobin-Haptoglobin and Sensitive Spectrophotometry. Biochem. J. 91, 161Complex. Biochim. biophys. Acta 100, 286-289. 171. NOBLE R. W., PENNELLYR. R. & RIGGS A. (1975) Studies GILLEN R. G. & RIGGS A. (1973) Structure and Function of the Functional Properties of the Hemoglobin from of the Isolated Hemoglobins of the American Eel, the Benthic Fish, Antimora rostrata. Comp. Biochem. Anguilla rostrata. J. Biol. Chem. 248, 1961-1969. Physiol. (In press). KOLM~RJ. A. (1949) Clinical Diagnosis by Laboratory Exa- RAYMOND S. (1962) A Convenient Apparatus for Vertical minations, 2nd Edn. Appleton-Century-Crofts, New Gel Electrophoresis. Clin. Chem. g, 455-470. York, p. 483. TAN A. L., NOBLER. W. & GIBSONQ. H. (1973) Conditions LAu H. K. F., WALLACHD. E., PENNELLYR. R. t~ NOBLE Restricting Allosteric Transitions in Carp Hemoglobin. R. W. (1975) Ligand Binding Properties of Hemoglobin J. Biol. Chem. 248, 2880-2888. 3 of the Trout, Salmo gairdneri: The Occurrence of an Acid Bohr Effect in the Absence of Heme-Heme Interaction. J. Biol. Chem. 250, 1400-1404. REFERENCES