The measurement of the Bohr effect of fish hemoglobins by gel electrofocusing

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THE M E A S U R E M E N T OF THE BOHR EFFECT OF FISH H E M O G L O B I N S BY GEL E L E C T R O F O C U S I N G * H. FRANKLIN BUNN I and AUSTEN RIGGS2 ~Department of Medicine, Peter Bent Brigham Hosp,al, Harvard Medical School, Boston, MA 02115, U.S.A.: -'Department of Zoology, University of Texas. Austin, TX 78712. U.S A (Reeeiced 5 April 1978)

Abstract 1. Hcmolysatcs from 16 species of Amazon lish and one amphibian were analyzed by gel clcctrofocusing. The change in isoclcctric point upon dcoxygcnation provided a reliable estimate of the Bohr effect. 2 Certain species of fish had single hemoglobin components whose p/ increased significantly upon dcoxygcnation, as in man. Other fish had hemoglobins whose tsoelcctric points were unaffected by deoxygenation Six species of fish had at least two hemoglobin components, one of which had a reduced isoelectric point upon deoxygenation indicating a reversed Bohr effect, whereas the other(s) had an increased moelectric point on deoxygenation, as occurs with the normal alkaline Bohr effect. 3. A close correlation was found between the change in moelecmc point with deoxygenation and the Bohr effect determined by oxygen equilibrium measurements. INTRODLICTION

showed a corresponding reduction in the difference between the isoelectric point (pl) of the liganded and unliganded forms (Jensen et al., 1975). In this paper, we extend this experimental approach to the study of fish hemoglobins. There is considerable heterogeneity in both the structural and functional properties of fish hemoglobins (Riggs, 1970: Bonaventura et al., 1975: Brunori, 1975). Some species have hemoglobins which lack heterotropic interactions while others contain hemoglobins with markedly exaggerated dependency of oxygenation on pH (the so-called Root effect). The physiological significance of the Bohr effect in fish may be broader than that of mammals. Hemoglobins with Root effects may help the unloading of oxygen into the swim bladder enabling fish to control their buoyancy (Riggs, 1970: Brunori, 1975).

The effect of pH on the oxygenation of hemoglobin, commonly called the "Bohr effect", continues to be of biochemical and physiological interest. Investigators near the turn of the century concluded that the Bohr effect not only facilitated transport of oxygen by hemoglobin to tissues but also enhanced the capacity of red cells to carry carbon dioxide to the lungs. The experimental and interpretive insights of W y m a n {1948, 1964)established this p h e n o m e n o n as a linked function and prototype of heterotropic interactions within allosteric proteins. The change of oxygen affinity with pH can be related to the differential affinity of oxygenated and deoxygenated hemoglobin for protons:

l ?pH

Jr

= [,;iH+q

.

L ? Y JP.

METHODS AND MATERIALS

The value of the left-hand expression can be experimentally determined by the measurement of oxygen equilibria at different pH, while the value of the righthand expression can be determined by acid-base titrations of solutions of oxyhemoglobin and deoxyhemoglobin. These determinations may not always be exactly equivalent, however, because the left-hand side of the equation is usually evaluated at half-saturation ( Y = 0 . 5 ) , whereas the acid-base titration measures the total difference in protons bound by deoxy- and oxyhemoglobin. A change m the affinity of protons for hemoglobin with oxygenation should be directly reflected in a difference m isoelectric point. The use of isolectric focusing in polyacrylamide gels has provided a quick method of sufficiently high resolution to measure the change in isoelectric point with deoxygenation (Park, 1973: Bunn & McDonough, 1974l. A h u m a n hemoglobin variant (Hb Syracuse ~2fl21,,3 ... -,,,,) with a reduced alkaline Bohr effect, * A

Specimens were obtained during an expedition on R.V. Alpha Helix" into the Amazon River basin. All samples were collected from fish m the waters within 50 miles of the joining of the Rio Solim6es and the Rio Negro or adjacent ponds and channels. Blood samples were obtained and hemolysates prepared as described by Fyhn et al. {1979). All gel focusing experiments were completed within 4 days after the preparation of the hemolysates. Hemoglobins were analyzed by isoelectnc focusing on polyacrylamtde gel, as described by Drysdale et al. (1971L Samples containing 100 fig of protein were applied to cylindrical gels {10 x 0.3 cm) containing 4",, acrylamide and 2",, amphohne pH 6 8 (LKB Produkter, Bromma. Sweden) Approximately 120min ',@re required for samples to reach their lsoelectric points. The gels were photographed and then stained with bromophenol blue. Since these experiments entailed analyses of deoxygenated hemoglobins, strict anaerobic conditmns were used. A solution of sodium ditluonite (5 mg/ml) was prepared by chasing deoxygenated water into the dry salt under positive nitrogen pressure. During the 20 mm period of prefocusmg prior to appIicatmn of samples, nitrogen gas was gently bubbled into the catholytc and 0.02 ml of the dlthmnite solution was applied to the top of each

Portuguese translation of this work will appear in

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95

H. FRANKLIN BUNN and AUSTEN RI(;GS

96

cylinder. This was effective m rernovmg the last traces of oxygen from the gels. Hemoglobin samples were dduted to a concentration of 10mg/ml m 0.05M bis-Tris [2,2-bis (hydroxymethyl)2,2',2"-nitriloethanol], pH 70. Samples (0.2 ml) were placed in vmls capped with a rubber vaccine stopper and deoxygen:ttcd by flushing with water-saturated nitrogen. Add~tton of 0.02 ml of the dlthlonite solution insured complete deoxygenatlon. A duplicate vml was gassed with carbon monoxide. For each hemolysate tested, three successive gels were loaded with deoxyhemoglobm, carboxyhemoglobin and a m~xture containing equal amounts of each. The pH gradient that was established under the above conditions was measured as described by Drysdule et al. (1971) Gel slices ( ~ 2 m m ) were placed in 0,3ml of 0.01 M KCI and thoroughly minced The following day the tubes were centrifuged and the pH of each supernate was measured at 5 C on a Radiometer pH meter with a capillary electrode These results confirmed the presence of a linear gradient from pH 60 to 7.8. The isoelectnc points of the hemoglobin bands were estimated by their measured positions in the gels compared with those of human carboxy and deoxy hemoglobins.

Table 1 Gel electrofocusmg patterns of various fish hemoglobins Hemoglobin Pattern

Effect of Deoxygenat~on on the Isoelectrtc Point

Single (or major) component

Two or more components

Increase

Human Osteoglossum Arapaima* Prochilodus Pseudodoras Xenocaru*

No change

Typhlonectes Lepidosiren* Ageneiosus

Increase

Spotted Ray*

No change

Doras Auchenipterus

Incrcase and Decrease

Hemiodus Mylossoma Rhytlodus* Hoplosternum Pimelodus* Pterygoplict hys

RF,SU LTS Sixteen species of fish and one a m p h i b i a n (a caecihan, Typhhmectes compre.~sicaudu) were examined. The results are summarized in Table 1. As shown in Fig. 1, some fish had patterns similar to that of the h u m a n hemolysate: a single major component whose isoelectric point increased significantly upon deoxygenation. A much smaller increase was observed in the Pseudodoras hemolysate. Several species tested had patterns for deoxyhemoglobm which were indistinguishable from those for carboxyhemoglobm (Fig. 2). These included animals with single hemoglobin components such as Tj'phhmecte,s and Ageneiosus as well as Lepido.siren (not shown). Many of the fish that were examined have multiple hemoglobin components. In two species, the Doras and Auchenipteru.s (Fig. 2), none of the hemoglobin components demonstrated any detectable shift in isoelectric point upon deoxygenation. In contrast, six other species of fish with multiple hemoglobin components had more complex patterns (Table 1). [n four species shown in Fig. 3, one or more hemoglobins whose pl increased upon deoxygenahon coexisted with a hemoglobin component of higher isoelectric point, which decreased upon deoxygenation. These include the Hoplostermtm and the Ptery,qoplichthys, the hemoglobins of which have been studied in detail by Garhck et ul. (1979) and Brunori et al. (1979) as well as those of Hemiodus and Mj'lossomu. Of the 18 different hemoglobins that wcrc analyzed by gel electrofocusing (Table 1), oxygen equilibria were determined on 12.* As Fig. 4 shows, we noted an excellent correlation between the estimate of the Bohr effect from change in pl on deoxygenation and the direct determination of the Bohr effect by measuring the oxygen affinity at different pH values. These data include the two purilied components from Hoplo.stermml (Garlick et al., 1979) and two from Pterygophchthys (Brunori et al., 1979). The reciprocal of the slope of the hne in Fig. 4 gives the approximate * Reported elsewhere m this issue

* Not shown

m

Orgamsm

figures.

number of protons released by either oxy- or deoxyhemoglobin upon unit increase in pH in the vicinity of the isoelectric point. This number, about eight pertetramer, is the same as the value of eight found for horse C O - h e m o g l o b i n (Cohn et al., 1937) and suggests that all of these hemoglobins have rather similar titration curves near their isoelectric points. In one experiment, we investigated the effect of temperature on the gel focusing patterns of deoxyand carboxy-hemoglobms. The four animals examined at 15 C (human, HoplostermmL Prochilodus and Hemiodus) had patterns indistinguishable from those obtained at the usual temperature 15 C). DISCUSSION

These experiments offer a new way of comparing the functional properties of various hemoglobins. The effect of deoxygenation upon the isoelectric point of individual hemoglobin components provides an indirect but accurate assessment of the Bohr effect. This approach is particularly useful in assessing the Bohr effect of fish hemoglobins since this p h e n o m e n o n appears to be more varied in these animals, m comparison to that in mammals. Furthermore, the fact that fish hemoglobins are often multiple makes the interpretation of the oxygen equilibria of the whole hemolysate difficult. The unexpected finding of a decrease in pl upon deoxygenation in a third of the species tested suggests that the presence of a ~'reverse'" Bohr effect (Alog Ps0/ApH > 0) may be more c o m m o n than previously thought. The presence of two hemoglobin components in Hoph~.sternum with opposite Bohr effects was confirmed by Garlick et al. (1979), who isolated the components and measured their oxygen equilibria. In like manner, isolated components of Pteryyophchthy~ demonstrated a close correlation between the shift in isoelectric point and the

Measurement or the Bohr effect of fish hemoglobins

Human

Deo CO

CO

Osteoglossum

Prochilodus

CO Deo CO

CO Deo CO

+

Deo

Pseudodoros

CO

Deo CO

-t-

+

Deo

Deo

+

Deo

97

Fig. 1. Gel electrofocusing patterns of hemoglobins of man, Osteoglossum, Prochilodus and Pse,dodoras. The first three had a more marked increase in pl upon deoxygenation compared to Pseudodoras.

Typhlonectes

Auchenipterus

Doras

Ageneiosus

"" ""I .

t

t i

k~

'ii

/'

? !', ;', "{~ :.., --,~ ~,~

~11~i ,~llb~ =I~ ~,I

CO Deo CO +

Deo

': ....

CO Deo co -i-

Deo

:4!~

-.

s.~! ~ .;~

-(',N

|

CO Deo CO +

Deo

CO Deo CO +

Deo

Fig. 2. Gel electrofocusing patterns of hemoglobins of Typhhmectes, Ageneios,s, Dortts and Auchenipteros, showing no change in pl upon deoxygenation.

98

H . FRANKLIN BUNN a n d AUSTEN RIGGS

Hemiodus

Hoplosternum

Ptery! loplichthys

Mylossomo

t

t

1

CO

Deo CO +

CO Deo CO

+

CO Deo CO +

CO Deo CO +

Deo

Deo

Deo

Deo

Fig. 3. Gel electrofocusing patterns of hemoglobins of Hoplo.stermmL Hemiodu.s, Pter.'t,lopichth.'s and M.l'lossoma showing a sigmficant decreasc in the pl of the cathodal hemoglobin component upon dcoxygenation. measured Bohr effect. It may be significant that all components demonstrating this "reverse" Bohr effect had relatively high isoelectric points. Glllen & Riggs (19737 and Weber et al. (1975) have observed such a "reverse" Bohr effect in cathodal components from two species of eel (Antjuilla rostrata and Anguilla an quilla). Reversed Bohr effects have also been found in the hemoglobins of certain Amphibia (Watt & Riggs, 1975: Bonaventura et aL, 1977). It will be of considerable interest to determine which amino acid residues are responsible for this property. Physiological interpretation of these gel focusing data must be viewed cautiously. Our results provide estimates of the Bohr effect only at the isoelectric point of the hemoglobin component. It is possible that the Bohr effect could be quite different at a physiological pH far removed from the isoelectric point. Furthermore, our data were obtained in the absence of erythrocyte organic phosphates which have profound effects both on the oxygen affinity and the pH dependence of oxygenation of fish hemoglobins (Gillen & Riggs, 1971: Powers, 1972). Interestingly, the "reverse'" Bohr effect of the cathodal eel hemoglobin was abolished by the addition of ATP (Gillen & Riggs, 1973). Finally, the gel focusing data were obtained at a single, non-physiological temperature (5' C). A significant effect of temperature on the Bohr effect of mammalian hemoglobins has been noted (Rossi et al., 1963: Antonini et al., 1965). Fish hemoglobins differ markedly from mammalian hemoglobin in the degree to which the liganded tetramer dissociates into dimers:

~2fl2

~ ~"

2~fl.

The hemoglobins of most mammals including man

readily dissociate into ctfl dimers under physiological conditions ( K 4 , 2 = 4 x 10 -6 M) (Gray, 1974). For this reason, when two liganded hemoglobins of unlike charge [i.e. human Hb A (~(2fl6c'h') and Hb S (~2f126v")] are mixed, half of the molecules in the solution will be in the form of the asymmetrical hybrid 06

~ ' 04

,,~ 02 k._

~ ~

o -02 -04

,

-i 5

i

~

I

I

-I 0

~

i

~

L

I

i

i

i

,

-05

I

0

,

,

,

,

I

5

/I Log ,°5o/L~ ply Fig. 4. Correlation or the Bohr effect estimated from gel electrofocusmg data (pl d e o x y - pl carbosy) with that determined by oxygen equilibria (Alog Pso/Alog pH) 0, fish hemoglobins: A,, normal human hemoglobin: /x human hemoglobin Syracuse (fl143 His---* Pro): II, amphibian hemoglobin (Typhlonectes). The Alog Psn/Alog pH was calculated at the pl of the hemoglobin component. I. Prochilodus. 2. Pterytjoplichthys (anodal component), 3. Pterygoplichthys (cathodal component), 4. Hoplosternum (anodal component), 5. Hoplomerum (cathodal component), 6. Osteo.qlossum. 7. Arapamltt, 8. Mylossoma (anodal component), 9. Pseudodoras, 10. Lepidosiren. The isoelectric points were determined at 4 C while the oxygen equilibria were done at 20 C.

Measurement of the Bohr effect of fish hemoglobins tetramer :t2flxfl ". Tiffs hybrid can be readily demonstrated by electrophoresis under anaerobic conditions after the hemoglobins have been stabilized by deoxygenatlon (Bunn & McDonough, 1974). In contrast, the liganded forms of some and perhaps all fish hemoglobins are much more stable tetramers (Edelstein et al., 1976). Tile K,,.2 for the two major components of trout oxyhemoglobin (Sahno irideus) are 7.5 × 10 -8 and 5.2 x 1 0 - S M (Brunori, 1975). For this reason, stable asymmetrical hybrids may exist in oxygenated hemolysates, and contribute to the multiple banding that is found among so many species of fish (Riggs, 1970). If so, the formation of additional hybrid tetramers may not occur following deoxygenatlon. We did not observe the emergence of asymmetrical hybrids in any of our experiments which were performed under conditions which readily allow the demonstration of such hybrids in mammalian hemoglobins. In summary, a good estimate of the Bohr effect of hemoglobin components of both fish and man can be obtained by isoelectric focusing. The method is much less time consuming than measurement of oxygen equilibria and can be readily done on hemolysates without the purification of the components. This approach lends itself to the survey of a wide variety of animal hemoglobins. .4tl, m~wle,lgement.~ This work was performed aboard R.V. Alpha Helix', and was supported by Grant PCM75-06451 from tile National Science Foundation, and by the Nehemias Gorm Foundation Additional support provided by NIH grant HL 16927 (HFB). NSF grant PCM-76-06719 (A.R.), and by the University of Texas Research Insmutc (A R ).

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99

BUNN H. F. & MCDONOUGII M. 11974) Asymmetrical hemoglobin hybrids: an approach to the study of subumt interactions. Biochemistry 13, 988-993. COHN E. J., GREEN A. A, & BLANCHARO M. H. (1937) Studies in the pysical chemistry of proteins XIV. The amphoteric properties of hemoglobin, d. Am. chem. Soe. 59, 509 517. DRYSDALE J. W., RIGHETTI P. & BUNN H F (1971) The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamlde gel. Bioelnm. hiophvs. Aeta 229, 42-50. EDELSTEm S. J., MCEwEN B. & GmsoN Q. H. (1976) Subunit dissocmtlon in fish hemoglobins, d hud. Chem. 251, 7632 7637. FYHN U. E. H., FYHN H J., DAWS B. J., POWERS D. A., FINE W. L. & GARLICK R. L. (1979) Hemoglobin heterogeneity in Amazonian fishes Comp. Bmehem. Phv.~iol. 62, 39 66. GARLICK R. L BUNN H. F., FYHN H. J., FYHN U. E. H, MARTIN J. P., NOBLE R. W & POWI:RS D. A. (1979) Functional studies on the separated hemoglobin conlponents of an air-breathing catfish, Hoplo.~termml littm'ah' (Hancock). Comp. Biochem. Phl'.~tol. 62, 219 226. G H-LIN R. G & RIGGS A. (I 971 )Thc hemoglobins of a fresh water telcost, Ciehla,soma evanoguttutnm 1. The effects of phosphorylated organic compounds upon thc oxygen equilibria. Comp. Biochem. Phv.~iol 38B, 585 595. GILLEN R. G. & RIGGS A. (1973) Structure and functmn of the isolated hemoglobins of the American eel Angullla rostrata. J. htol. Chem 248, 1961 1969. GRAY R. D. 11974) The effect of 2, 3-diphosphoglycerate on the tetramer-dimer equihbrmm of hganded hemoglobin. J, bud. Chem. 249, 2879-2885. JENSEN M., OSKi F A, NATHAN D. G. & BUNN H. F. 11975) Hemoglobin Syracuse (~,//., 14,3(112111h, o lho), a new high-affinUy varmnt detected by specml electrophorettc methods. J. ehn. Im'e,st. 55, 469 477 PARK C. M. (1973) Isoelectrlc focusing and the study of interacting protein systems: hgand binding, phosphate binding and subunit exchange in hemoglobin..4mL N.Y. Acad. Sci. 209, 237-257. POWERS D. A. 11972) Hemoglobin adaptation for fast and slow water habitats m sympatnc catostomld fishes. Scwnee 177, 360-362 RIGGS A. (1970) Properties of tish hemoglobins. In Ft.ql Phv.smloqv. Vol. 4 (Edited by HOAR W. S. & RANDALL D. S.), pp. 209 252. Academic Press, New York. Ross] L., CHIPPI:RI-IFLDJ. R. & ROUGH'ION F. J W. 11963) The effect of temperature on the titration curves of human oxygenated and reduced haemoglobin. Bmehem. J. 87, 33. WAIT K. W K. & RIGGS A. 11975) Hemoglobins of the tadpole of the bullfrog, Rtma eate.q, emna. Structure and function of the isolated components d. hiol Chem. 250, 5934~5944 WEBER R E, LYKKI'BOI- G. & JDHANSEN K (1975) Physmlogical properties of eel hemoglobin' hypoxlc accllmahon, phosphate effects and multiplicity J. exp. Bud. 64, 75 88. WYMAN J. (1948) Heine proteins..4dr. Protein Chem. 4, 407 531. WYMAN J. (1964) Linked functmns and reciprocal effects in hemoglobin. A second look. Adr. Protein Chem. 19, 223-286