0300.Y629!X3,030545-04$03.00~0 c 1983 Pergamon Prcas Ltd

COMPARATIVE STUDIES OF HEMOGLOBINS FROM NEWTS (TRITURUS CRISTATUS, TRITURUS

VULGARIS, TRZTURUS ALPESTRIS): A KINETIC APPROACH

SAVERI~ G. CONDO)*, BRUNO GIARDINA~, ANDREA BELLELLI*, MARIO LUNADEI?, ALESSANDRO FERRACIN~ and MAURIZIO BRUNORI*

*Institute of Chemistry. Faculty of Medicine and Consiglio Nazionale delle Ricerche, Center of Molecular Biology, University of Rome

iInstitute of General Biology, Policlinico Umberto I. Rome $Institute of Biochemistry, Faculty of Science, University of Cagliari

(Receirrd 9 July 1982)

Abstract-l. The functional properties of three different species of newts i.e. Triturus cri.status ~UTIII‘~C.Y. Triturus culgaris meridiona/i.s and Triturus alpestris apuunus have been studied by flash photolysis.

2. In spite of the very different habitats the hemoglobins of the adult form of the three species do not show significant differences in functional behaviour.

3. However the neotenic form of Triturus alpestris apuanus is characterized by the presence of a higher multiplicity of hemoglobin components as compared to that of the adult form.

4. The experiments show that the new hemoglobin(s) is characterized by a more marked pH depen- dence and by a much higher rate constant for CO combination.

5. This finding may be relevant to the compulsory aquatic life of the neotenic form of this amphibian.

INTRODUCTION

The functional properties of hemoglobins from Tri- turm cristutus have been recently characterized both from equilibrium and kinetic point of view (Condb et

al., 1981). The results indicated that both the ampli- tude and the shape of the Bohr effect are dependent on the presence of organic phosphates, providing a fine modulation of O2 transport in relation to the physiological demands. The experimental data, obtained during this study, have shown a satisfactory agreement between O2 equilibria and CO kinetics. This makes it possible to explore kinetically the func- tional properties of hemoglobins from several species of newts in order to underscore possible functional differences induced by the specific characteristics of the living environment. The kinetic approach, in fact allows us to obtain and compare rapidly some of the main functional characteristics (i.e. ligand reactivity, ligand-linked conformational changes, etc.) of differ- ent hemoglobin systems even when, as in the present case, very small amounts of material are obtainable for difficulties related to availability and body size of the animals. This note reports a comparative kinetic study (flash photolysis) performed on three different species i.e. Triturus cristatus carnrjex, Triturus vulgaris meridionalis and Triturus alpestris apuanus. Further- more, in the last case (Triturus alpestris) we have par- tially characterized also the hemoglobin system of the neotenic form, in order to investigate possible effects of metamorphosis that is of the transition from aqua- tic to terrestrial life. In fact it is well known that during metamorphosis amphibians exhibit modifica- tions of their hemoglobins (Trader & Frieden 1966; Brown & Dewith. 1970: Cardellini et al.. 1978; Flavin rt al., 1978).

MATERIALS AND METHODS

Animals were collected from ponds near Rome (Triturus. cristatus carnifu and Trituws- wlguris meridionalis) and from Bega lake (Lucca, Central Italv) in the case of Tri- turus al;stris apuanus specimens. Blood has been washed three times in isotonic (0.7% NaCI) solution containing 2 mM EDTA and then the packed cells have been lysed by adding two volumes of cold distilled water.

Stroma and nuclei were removed by centrifugation for 30 min at 10,000 rpm. Analytical isoelectric focusing was performed on polyacrylamide gels as described by Drys- dale et al. (197 1). Samples containing 50 gg of protein were applied to the gels (IO x 0.3 cm) containing 42, acrylamide and 2% ampholine pH 68 (LKB, Sweden). The run has been performed at 300 V for 4 hr at 4‘C. Flash photolysis experiments were performed by means of an apparatus similar to that described by Brunori & Giacometti (1981). Spectrophotometric measurements were carried out with a Cary 219 spectrophotometer. Carbon monoxide was obtained from Caracciolo (Rome). All other chemicals were of chemical grade.

RESULTS AND DISCUSSION

The adult stage of the three species As already observed in the case of Triturus cristatus

carnlJex (Condb et al., 1981) electrophoretic analysis of the hemolysate from single animals (at least 5 indi- viduals for each species) shows the presence of mul- tiple hemoglobin components.

Moreover the species-specific electrophoretic pat- tern shows in all three cases, a significant degree of individual variability as to the relative proportion of hemoglobin components. However, in the case of Tri- turus cristutus carn[f& (Condb et al., 1981) a set of flash photolysis kinetic experiments, performed in parallel on the purified main Hb component and on

545

546 SAVERIO G. CONDO rt al.

Jr p” 60

. 0

. D

pH 86

time (msec)

Fig. 1. Effect of pH on the time course of CO combination with hemoglobins from Triturus cristatus after full (open squares) and partial (closed squares) (20?,, photodissociation) photolysis in the presence of P,-inositol in saturating amounts (2 mM). Bis-Tris or Tris-HCI buffer 0.1 M + NaCl 0.1 M; Hb concentration = 4 PM; CO concentration = 44 /cM ; observation wavelength = 436 nm.

Temperature = 20 C.

the whole hemolysate, showed no detectable differ- ences in the kinetic behaviour. This finding was taken as an indication of kinetic homogeneity and therefore the functional characterization was carried out with the hemolysate. On the basis of a similar assumption we carried out these new experiments using whole hemolysates.

The present kinetic characterization was performed as a function of pH and in the presence of saturating concentration (2 mM) of P,-inositol.

The time course of CO recombination is shown in Figs 1, 2 and 3A and the relevant rate constants are reported in Table 1. The overall kinetic behaviour is very similar in the three cases and may be summar- ized as follows:

(i) The autocatalytic character of the progress curve, i.e. the kinetic expression of cooperativity in ligand binding (Antonini & Brunori, 1971), tends to vanish towards acid pH values. In particular at pH 6.0 the CO combination corresponds to pseudo- ‘first order kinetics, in complete agreement with the non-cooperative Oa binding observed previously at equilibrium in the case of Triturus cristatus.

(ii) Upon complete photolysis, there is no evidence for a quickly reacting kinetic component, which in the case of human Hb has been attributed to the pres- ence of @ dimers (Antonini & Brunori, 1971). Thus even in the liganded derivative newt hemoglobins show no tendency to dissociate into dimers and are tetrameric down to micromolar concentrations,

3r r

(iii) Upon partial photolysis (-20% photodissocia- tion), at pH 7.5 and 8.6, carbon monoxide recombina- tion is much faster (- 10 times) than that measured after complete photodissociation. This effect, which is a clear indication of the presence of ligand induced conformational changes, is almost negligible at pH 6.0 in agreement with the pseudo-first order kinetics ob- served at this pH and with the interpretation pre- viously reported (Condb et al., 1981) (i.e. that saturat- ing P,-inositol stabilizes newt hemoglobins in a low affinity quaternary state thereby abolishing the struc- tural basis of cooperativity).

(iv) The effect of pH on the kinetic constant for carbon monoxide binding is quite small (see Table 1), suggesting a major role of the “off kinetic constants as the change of ligand affinity with pH is concerned.

It may be concluded that, in spite of the different habitats, the hemoglobins of the adult form of the three species do not show significant differences in functional behaviour. In this respect it may be useful to recall that the control mechanisms developed by newt hemoglobins enable animals to vary the ampli- tude, the shape and the sign of the Bohr effect. This provides a fine modulation of 0, transport without changes involving the intrinsic characteristics of the proteins.

n?e neotenic form of Triturus alpestris apuanus

Electrophoretic patterns of the hemolysate from either the neotenic (Cohen, 1977) or the adult form

time (msec)

Fig. 2. Effect of pH on the time course of CO combination with hemoglobins from Trituru.5 cxifgaris. For symbols and conditions see Fig. I.

: r .

. 0

0 .

. D

.

. r

. or . o- oc

oL 100 200

I . D

50 100

547

time (msec)

Fig. 3. Effect of pH on the time course of CO combination with hemoglobins from Triturus alpestris (A) in the adult form and (B) in the neotenic one after full (open squares) and partial (20%) photolysis (closed squares). Adult form: hemoglobin 4 PM, CO 44pM. Neotenic form: hemoglobin 4pM, CO 100,~M.

Other conditions as reported in Fig. 1.

are shown in Fig. 4. At least two additional hemo- globin components, accounting for more or less 507; of the total pigment, are present in the neotenic form of Triturus alpestris apuanus. The presence of these two hemoglobins has been observed in all the eight individuals examined, except one (which lacks a com- ponent present in all other individuals).

Figure 3B reports the time course of CO recombi- nation for the neotenic form as a function of pH in the presence of saturating amount of P,-inositol. At pH 7.5 a large effect of partial photolysis is observ- able. Moreover, the time course corresponds to a simple kinetic behavior with a second order kinetic

constant very similar to that obtained for the adult animal (see Table 2). The autocatalytic character of the progress curve, observed at this pH in the case of adult T. ulpestris. is no more evident for the neotonic hemoglobin system suggesting a small degree of kin- etic heterogeneity.

That this is the case is demonstrated by the experi- ments performed at more acid and alkaline pHs.

Thus, both at pH 6.0 and 8.6. there is the appear- ance of a fast kinetic component which accounts for -25:; of the total optical density change and which contributes to mask the effect of partial photodisso- ciation (especially at pH 8.6). A set of experiments

Table 1. Etlect of pH on the apparent second order velocity con- stants for the combination of hemoglobins from the various species of

Triturus with carbon monoxide

Species PH k/M per set k*!M per set

Triturus cristatus 6 7.0 x lo4 2.2 x 10” 7.5 1.3 x 105 I.2 x 106 8.6 1.3 x 105 1.X x 10”

Tritwm u1pe.stri.s 6 6.3 x IO“ 7.5 7 x 10” X.6 1.0 x IO’

Triturus ~~lguris 6 3.5 x lo4 7.5 7.5 x lo4 8.6 1.0 x lo5

1.2 x IO’ 1.0 x 10” I.0 x 10”

3.4 x 10’ 1.1 x 10” 1.5 x 10”

k is the initial rate constant calculated from the time course corre- sponding to full photodissociation. k* represents the rate constant of the quickly reacting form observable upon partial photodissociation (ZO”,,). Bis-Tris or Tris buffer 0.1 M + NaCl 0.1 M in the presence of 2 mM P,-inosltol.

548 SAVERIO G. CONDO et al.

b a Fig. 4. Analytical isoelectric focusing of hemoglobins from Triturus alpestris (a) adult and (b) neotenic. pH gradient 8.0

(top) to 6.0 (bottom).

performed as a function of hemoglobin concentration (from 5 to 0.5 PM) at pH 6 has allowed to exclude a significant presence of dimers even for the neotenic hemoglobin system, since the relative proportion of the fast and slow phase remains unchanged. Therefore we may tentatively ascribe the fast kinetic component

Table 2. Apparent second order velocity constants for combination of CO with hemoglobins from Triturus alpes-

tris in the neotenic and adult form

PH Adult form k/M per set

Neotenic form Slow phase Fast phase k/M per set k/M per set

6 6.3 x 10“ 8.5 x 10“ 7.8 x IO5 7.5 7.0 x lo4 1.1 x lo5 8.6 1.0 x lo5 1.5 x lo5 1.0 x lo6

Photodissociation: 100%; Bis-Tris or Tris buffer 0.1 M + NaCl 0.1 M in the presence of 2 mM P,-inositol.

to the presence of the new hemoglobin band(s) ob- served in the polyacrylamide gel. From the experi- ments it results that the new hemoglobin(s) is charac- terized by a more marked pH dependence (as evident from the values of the second order kinetic constants reported in Table 2) and by a much higher rate con- stant for CO combination. It should be remarked that the absolute values of the second order kinetic con- stants calculated for the slow kinetic component observable in the case of neotenic 7: alpestris a. at pH 6.0 and 8.6, are very similar to those obtained in the case of the adult animal upon complete photodis- sociation. Therefore, although these characteristics are not immediately correlated to the different physi- ology of the animals before and after metamorphosis they seem to indicate the presence of hemoglobin(s) characterized by higher ligand reactivity and a more marked kinetic Bohr effect. Considering the compul- sory aquatic life of the neotenic form this is not com- pletely irrelevant.

REFERENCES

ANTONINI E. & BRUNORI M. (1971) Hemoglobin and Myo- globin in their Reactions with Ligands. North Holland, Amsterdam.

BROWN R. E. & DEWITH W. (1970) Hemoglobin changes during metamorphosis in Triturus uiridiscens. Comp. Bio- them. Physiol. 35, 495-497.

BRUNORI M. & GIACOMETTI G. M. (1981) Photochemistry of hemoglobins. M&h. Enzymol. 76, 582-595.

CARDELLINI P., GABRION J. & SALA M. (1978) Electro- phoretic patterns of larval, neotenic and adult hemo- globin of Triturus haelreticus. Ra:. Acta Embr. Exp. 2, 151L161.

COHEK J. (1977) Reproduction. Butterworths, London. CONDU 3. G.. GIARDINA B., LUNADEI M., FERRACIN A. &

BRUNORI M. (1981) Functional properties of hemo- globins from Triturus cristatus. Eur. J. Biochem. 120, 323-327.

FLAVIN M., BLOIJQUIT J., DUPRAT A. M. & ROSA J. (1978) Biochemical studies of the hemoglobin switch during metamorphosis in the salamander Pleurodeles waltli. 11. Comparative studies of larval and adult hemoglobins. Comp. Biochem. Physiol. 61B, 539-544.

DRYSDALE J. W., RICHET~I P. & BUNN H. F. (1971) The separation of human and animal hemoglobins by iso- electric focusing in polyacrylamide gel. Biochim. hiophys. Acta 229, 42--50.

TRADER C. D. & FRIEDEN E. (1966) Dimeriration and other chemical changes in amphibian hemoglobins during metamorphosis. J. biol. Chem. 241, 357-366.