Oxygen binding by Callianassa californiensis ... - ACS Publications

Physiol. 39B, 855. Tanford, C. (1961), Physical Chemistry of Macromolecules,. New York, N. Y., Wiley, Chapter 4. Thompson, R., and Prichard, A. W. (19...
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MILLER

Kegeles, G., Rhodes, I . , and Bethune, J. L. (1967), Proc. Ncir. Acrid. Sci. U.S. 58,45. Kirkwood, J. G. (1954),J. Polj.m. Sci. 12, 1. Loehr, J . S., and Mason, H. S. (1973), Bioclieni. Biopli>~s.Res. Coiiimun. 54, 741. Long, C.: Ed. (1961), Biochemists Handbook, Princeton, N. J.. van Nostrand, p 28. Matthews, B., and Bernhardt, S. (1973). Annu. Rer. Bioplij~s. Biomg. 2,257. Mil1c.r. K., and Van Holde, K. E. (1974), Biocheruisir~,13. 1668. Nickerson, K. W . , and Van Holde, K. E. (1971), Conip.

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V A N

HOLDE

Binclietn. Ph ysiol. 39B, 855. Tanford, C. (1961), Physical Chemistry of Macromolecules, New York, N . Y . ,Wiley, Chapter 4. Thompson, R., and Prichard, A. W. (1969), Biol. Bull. 136, 114, 274. Van Holde, K . E. (1974), Proteins (in press). Van Holde. K . E., and Cohen, L. B. (19641, Biocliemictry 3, 1803. Van Holde, K. E., and van Bruggen, E. F. J. (1971), in Subunits in Biological Systems, Timasheff, S. H., and Fasman, G. D., Ed., New York, N. Y . ,Marcel Dekker. Yphantis. D. A , (1964), Biochemisfrj?3, 297.

Oxygen Binding by Callianassa calzforniensis Hemocyanini Karen Miller and K. E. Van Holde*

.\RCTK,ICT: The oxygen binding by the hemocyanin of the ghost shrimp, Culliunussu culi/orniensis, has been studied as a function of pH, divalent ion concentration, and temperature. Most of the experiments were performed with the hemocyanin C component. which is competent to undergo an association from a 17s form to a 39s tetramer near neutral pH. It is demonstrated that the effects of pH and divalent ions on oxygen binding can be described by the theory of Monod et u I . ( d . Mol. Biol. 12, 88 (1965)) for allosteric transitions as

A

s has been described in the prcceeding paper (Roxby

1974), the ghost shrimp Culliunussu culiforniensis has a hcmocyanin capable of a completely reversible monomertctramcr association. There exist normally in the blood two components which can be completely separated on a ASm column. One sediments as a 17s component in the ultracentrifuge and the other as a 3% component.' The 39s component shows the recersible dissociation, breaking down into 17s subunits when Mg'' is removed, and reassociating to 39s subunits when Mg2' is replaced to a level of 0.05 M or greater. Wt. have designated this hemocyanin C. The 17s component nornially present in whole blood is incompetant to associate into 39s subunits regardless of the MgYi 1 hcmocyanin I. At high pH 1 2 9 . 2 ) the hemocyanin dissociates further into 5s subunits, which apparently rcpresent individual polypcptide chains. We h a w shown that the 17s component is a hexamcr of these subunits. (Roxby c'i d., 1974). Wc wished to investigate oxygen binding by Ciilliuncissii hemocyanin for a number of reasons. The background respiratory physiology of this shrimp has been investigated (Thompson and Pritchard, 1969; K . Miller and Pritchard, in preparation) and in particular preliminary measurements P I (ti.,

modifled by Buc et ul. ( J . Mol. Biol. 76, 199 (1973)). The 17s hexamer of polypeptide chains appears to be the allosteric unit. The T and R states of this 17s particle exhibit differing tendencies to associate. The 17s particles of hemocyanin I, which are not capable of association, have a T state identical with that of hemocyanin C , but cannot adopt the same R state. Possible significance of this behavior to the physiology of the shrimp is discussed.

were made of the oxygen binding of whole blood. It seemed logical in this case to attempt to duplicate the conditions of divalent ion composition, temperature, and p H normally found in whole blood under completely controlled conditions in an attempt to fully understand the effect that varying physiological states might have on the oxygen binding properties of the hemocyanin. The normal variations in physiological pH have been measured (K. Miller and Pritchard, in preparation). Several researchers report a pronounced effect of Ca?' ions on oxygen binding in other hemocyanins (Hwang and Fung, 1970; Larimer and Riggs, 1964) and Mg?' ions have been shown to have the same effect (Larimer and Riggs, 1964). The blood calcium levels in Culliunussu have been measured by Thompson and Pritchard (1969) but it was necessary for us to measure the norinal hemolymph Mg2' levels before composing a buffer system for dilute samples. Since with this hemocyanin one can obtain several different stable aggregation states, as well as some equilibrium mixtures of these, it makes an ideal system for the study of the interrelation between aggregation state and oxygen binding. In this paper we describe binding studies with both hemocyanin C and hemocyanin I, as a function of pH, divalent ion concentration, and temperature. Experimental Section Preparation of Suluiions. Shrimp were dug at Yaquina Bay and bled, and the blood was purified on a Bio-Gel A5m column as described previously (Roxby et al., 1974). The eluent for the column was 0.1 I Tris buffer (pH 7.65) with

OXYGEN BINDING BY

Callianassa

HEMOCYANIN

0.05 M MgCI2. Unless otherwise specified all experiments were done with the purified 39s fractions, hemocyanin C. All solutions were made with 0.1 l buffers prepared according t o the Biochemists Handbook (Long, 1961), using doubly distilled water. For binding studies in which we wished to approximate the divalent ion composition of hemolymph, 0.05 M MgC12 and 0.01 M CaC12 were added, either directly or by dialysis, to the samples. In most cases dialysis was used, and was carried out overnight, with one replacement of the dialysate, using a volume ratio of dialysate to solution of about 1OO:l in each case. The 17s form of hemocyanin C was prepared by dialyzing the 39s fraction against Tris buffer without added Mgz+. The 5 s subunits were prepared by dialyzing the 39s fraction against pH 9.2 borate or bicarbonate buffer. Measurement of Mg2+. Mg2+ levels in whole blood diluted 1 :400 with distilled water were measured by atomic absorption spectroscopy using a Perkin-Elmer Model 303 spectrometer. To resolve the Mg2+, 1 % CaClz was added to the sample before flaming. Oxygen-Binding Studies. Measurement of oxygenation of the hemocyanin was based on the 337-nm absorbance band. This band is absent in the deoxygenated hemocyanin which exhibits only scattering at this wavelength; the band increase in absorbance ( A ) with increasing oxygenation. The tonometer consisted of a glass bulb of 107.2-ml volume with a 1-cm fused-quartz cuvet attached. The ground glass stopper of the tonometer was equipped with straight sidearm consisting of a small capillary between two stopcocks. Depending on the oxygen affinity of the solution used the volume of the capillary used was either 1.1819 or 0.1718 ml. The volumes of the sidearms were determined by mercury weighing and that of the tonometer by weighingit filled with water. A hemocyanin solution of Aaa5'v 1, corresponding to a protein concentration of 3-4 mg/ml, was placed in the tonometer and deoxygenated by alternate evacuation and nitrogen flushing until the peak at 337 nm was completely eliminated. Since it appeared likely that the oxygen binding might, under some circumstances, depend upon degree of association, and hence the concentration, all measurements were made with approximately the same hemocyanin concentration. Capillary increments of air or oxygen at 1 atm were added; the choice of gas depended on the oxygen affinity of the hemocyanin under the circumstances of the experiment. The tonometer was equilibrated in a temperature-controlled shaker bath for 10 min after each addition and the absorbance spectrum from 400 to 300 nm was quickly measured. Longer equilibrium did not increase the A33i.All binding curves, except for the temperature series, were made at 25 i 0.1". At low temperatures only the portion of the spectrum from 345 to 330 nm was measured, to prevent excessive changes in temperature from altering the absorbance readings. Gas was added until a pressure of 1 atm was reached. The oxygen partial pressure was calculated according to the following equation used by Spoek et al. (1964).

F = volume fraction of O?in the gas added: 0.2045 for air, 1.000 for oxygen; B = barometric pressure; W = water vapor pressure; pCOf = COz pressure negligible in our experiments; V = volume of tonometer; u = volume of sidearm; i = increments of gas added. In cases where the blood was not fully saturated at 1 atm of air, or 1 atm of 02,the curves were extrapolated to infinite

I 1

'

0

log PO,

1 : Hill plots of the binding of 0% by hemocyanin C at 25", pH 7.65. The numbers on the code indicate Mg*+ concentrations. The two straight lines with slope unity (T and R) are placed according to data in Figures 4 and 6. FIGURE

oxygen pressure by plotting 1/A us. l/pOz. The absorbance value at the y intercept ( A , ) was used for calculations of fraction saturation of the hemocyanin (7)

B

=

( A - Ao)/(A, - Ao)

(2)

where A . is the value of the residual absorbance due to light scattering by the deoxygenated hemocyanin. In the following sections, we shall present data in several ways. In some cases we will give binding curves as P us. PO*; in other cases Hill plots (log [B/(l - P)] us. log poi) will be used. In Tables we will present p50, the oxygen pressure at P = and n H , the maximum slope of the Hill plot. Sedinwntation Experiments. Sedimentation velocities were measured with the Spinco Model E analytical ultracentrifuge. Either the ultraviolet optical scanner or phase-plate schlieren optics were used. In experiments in which deoxygenated hemocyanin was used, the cell was loaded under nitrogen in a glove bag. Sedimentation coefficients were calculated either from the peak maximum (schlieren data) or from the halfmaximum absorbance (scanner data). All data were corrected to s ~ ~values. , , ~ Results We have investigated the effects of a number of parameters on the oxygen binding by Callianassa hemocyanin. These parameters include divalent ion concentration, pH, and temperature. These investigations were guided by two aims: (1) to elucidate the relationship (if any) between the state of aggregation of the hemocyanin and oxygen binding, and (2) to attempt to understand the significance of these parameters (pH, divalent ion concentration, and temperature) to the physiological function of the hemocyanin. Effects of Divalent Ions on Oxygen Binding. Our investigations of divalent ion effects on binding were first prompted by our observations (Roxby et al., 1974) that Mgz+ and Ca2+ markedly influenced the association equilibria. Others have noted divalent ion effects on both association and oxygen binding by hemocyanins (see, for example, Larimer and Riggs (1964) and Hwang and Fung (1970)) but to our knowledge no complete study of the interrelation of such effects has appeared. Accordingly, we carried out the series of experiments illustrated in Figure 1 and Table I, in which oxygen binding was measured at pH 7.65 in the presence of different concenB I O C H E M I S T R Y , VOL.

13,

NO.

8, 1 9 7 4

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MILLER AND V A N HOLDE

0 2 M Mg"

025 M Mg*+

04 M Mg'*

I/

/I '

I

2: Schlieren patterns of oxygenated and deoxygenated hemocyanin C at different Mg2+concentrations (as indicated). All data are at approximately 18". pH 7.6. Each photograph was taken after about 16-min ultracentrifugation at 40,wO rpm. The boundariescorrcspondtoapproximately 16and 35 s,respectively.

FIGURE

trations of MgZ+. Solutions were prepared by dialysis of puriSed hemocyanin C us. the appropriate buffers. Hemocyanin that has been dialyzed against Mg*+-free buffer is entirely in the 17s form (Roxby et a/., 1974) and exhibits a nearly noncooperative binding curve, and very weak binding overall (see Figure 1). As [Mg*+] is increased, the binding becomes stronger and the curve exhibits more and more cooperativity, as indicated by the maximum slope of the Hill plot. At Mg*+ concentration of 0.05 or greater, the protein is almost entirely tetrameric (almost completely in the 39s form) in both the oxygenated and deoxygenated states (see Figure 2). Raising [Mg*+lstill higher increases the binding strength (lowers p50). This series of experiments was arbitrarily terminated at [Mg'+] = 0.1 M; in retrospect (see Discussion) it would have been of interest to iontinue t o higher [Mg'+]. At low [Mg'+l(