Studies on the reaction of isocyanides with haemproteins

J. Mol. Biol. (1972) 65, 423-434

Studies on the Reaction of Isocyanides witi Haemproteins tII. Binding to Normal and Modified Human Haemoglobins MATJRIZIO BRUNOBI,BERNARD TALBOT,ALFREDO COLOSIMO ERALDO ANTONINI AND JEFKRIES WYMAN

In.stitute of Biochemistry, C.N.R. Centrefor Molecdur Biology University of Rome,and Regina Elena In&We for CancerResearch,Rome,Italy (Received13 September1971, and in revisedform 13 December1971) This paper reports the results of an investigation of the binding of methyl-, ethyl-, isopropyl-, n-propyl- and tertiary-butyl-isocyanides with normal human haemoglobin, meso- and deutero-reconstituted haemoglobins, several modified forms of the protein and the artificial CN-met intermediates. Consideration of the experimental data allows some conclusions to be drawn. (a) The functional properties of haemoglobin appear to be independent of the nature of the ligand, since both homotropic and heterotropic interactions are maintained, to a large extent, in the case of the bulky isocyanides. In addition, it is observed that any chemical modification which &hers the &lnity or the co-operativity in the resetion of hsemoglobin for oxygen, also results in a similar change in the resotion with any other ligand. (b) For normal haemoglobin the value of n in the Hill equation is uniformly lower for the isocyanides (2.4 to 2.2) than for oxygen or carbon monoxide under comparable conditions, and the value of the partition oonstant between isopropylisocyanide and oxygen is variable with the extent of the replacement. These facts may be rationalized on the basis of a functional heterogeneity between the LYand ,3 chains in their reactions with bulky ligands. A ilt of the experimental data indicates that in the sssembled haemoglobin molecule one of the two chains may have an afEnity three to four times higher than the other. (c) The over-all equilibrium constant (I=(C,,,)-l) for the binding of haemoglobin to the isocysnides depends on the molecular dimensions of the ligand in a complex manner. An explanation of the pattern can be provided on the basis of the knowledge that the aliphatic side-chain of the ligand tends to be pscked toward the interior of the molecule, in anon-polar environment. Therefore, binding would be favoured by the transfer of the hydrocarbon chain from water to the nonpolar environment, and hindered by steric effects which become more and more serious as the size of the ligand increases.

1. Introduction The equilibrium and kinetics of the reaction of various alkylisocyanides with the isolated a! and t9 chains of human haemoglobin have been reported in a previous paper (Talbot, Brunori, Antonini $ Wyman, 1971). The present paper deals with the equilibrium of alkylisocyamcles with normal human haemoglobin together with several modifications of it, namely: (1) those involving the substitution of protohaem by meso- or deutero-haem; (2) haemoglobin modified in the protein moiety, such as iodoacetamide-treated haemoglobin, oarboxy-peptidase Adigested haemoglobin or Hb Rainier, i.e. (~s!ls~r~ 145+cYs; (3) the artificial haemoglobin intermediates,

01+~~pand ++CN. t Paper I in this series is Talbot, Brunori, Antonini & Wymsn (1971). 423

424

ill.

BRUNORI

ET

AL.

The results reported below lead to conclusions of general interest. First of all, they yield a more complete picture of the relations between the functional properties of haemoglobin and the nature of the haem ligands. In addition, evidence is provided that, in the assembled molecule, the 01and /I chains are not equivalent in the binding of the more bulky ligands, in agreement with other data. Finally, the dependence of the free energy of binding on the length of the aliphatic side-chain in the various isocyanides is tentatively rationalized.

2. Materials and Methods The preparation of haemoglobin and the isolation of the (Yand fi chains were carried out as in previous studies; the reconstituted haemoglobins were made by mixing proto-, meso- or deutero-haem with an equivalent amount of globin (Antonini & Brunori, 1971). The artificial intermediates (LX+cNj3 and L$‘~~) were prepared as described previously (Brunori et al., 1970). The following modified haemoglobins were used : iodoacetamidetreated Hb (Taylor, Antonini, Brunori & Wyman, 1966) ; p-chloromerouribenzoateHb, prepared using 2 moles p-chloromercuribenzoate per tetramer (Taylor et al., 1966); carboxypeptidase A-digested Hb (Zito, Antonini & Wyman, 1964) lacking His 146 and Tyr 145 on the /I chains; haemoglobin Rainier. The latter was kindly provided in pure form by Dr K. H. Winterhalter (see Amiconi, Winterhalter, Antonini & Brunori, 1972, for the purification procedure). The source, storage, stability and method of determining the concentration of MeNC, EtNC, Pr*NC and ButNC have been reported in detail in a previous paper (Talbot et al., 1971). m-Propyl isocyanide was obtained from Schuchardt Co., Munich, Germany; it contained a red impurity which was soluble in water, and was extracted with water from the parent material each time before use. In all cases, equilibria were determined either directly, or by measuring the partition with oxygen, as described by Talbot et al. (1971). 3. (a) Spectral properties

Results

of Gocyanide derivatives

of haemoglobin

Table 1 reports the spectral characteristics of the deoxy and liganded forms of normal haemoglobin as well as of meso- and deutero-reconstituted haemoglobins in the Soret region. These deoxy values were used throughout the work to measure the protein concentration. It is seen from the Table that the spectral properties of the various isocyanide derivatives of a given haemprotein are closely similar (see also St George & Pauling, 1951); on the other hand, there is a wide variation in spectral properties for a given ligand as one passes from one haemprotein to another. (b) The reaction with normal huemoglobin as measured directly This reaction was studied by adding successive amounts of ligand to a buffered deoxygenated solution of haemoglobin containing a small amount of sodium dithionite, just as in the case of the study of the isolated chains (Talbot et al., 1971). Over the spectral range used (4.25 to 440 nm) and in the solvents used (0.2 M-phosphate, pH 7.0, or O-05 M-borate, pH 9-l), the spectra showed good isosbestic points in all cases (for MeNC, EtNC, Pr’NC, Pr”NC, the isosbestic points were, respectively, 436, 437, 436 and 435 nm). In contrast to this, Olson $ Gibson (1970) have reported, in the lower spectral region (420 to 423 nm), marked deviations from isosbesty during titration of haemoglobin with another isocyanide, i.e. Bu”NC, in the presence of organic phosphates (in particular with inositol hexaphosphate). Table 2 records the over-all equilibrium constant (I=(C’,,z)-l) and the Hill parameter (n) for the binding of five isocyanides to haemoglobin at neutral pH and 20°C.

REACTION

OF

ISOCYANIDES

WITH

HAEMPROTEINS

425

TABLE 1 Spectral properties of vxwmul human and rewndituted haemoglobin derivatives in tb range 440 to 400 nm in 0.2 M-pota8sium plwsphate, pH 7.0, 20°C e x 10-3 (ems/m-mole)

Derivative

EtNC Pr’NC PrnNC

430 427 428 428 429

133 186 193 186 178

Meso-reconstituted haomoglobin

Deoxy MeNC EtNC Pr*NC

421 417 417 418

115 234 230 228

Deutero-reconstituted haemoglobin

Deoxy MeNC EtNC Pr’NC

421 416 419 417

115 203 204 196

Haemoglobin

Deoxy

MeNC

Each entry in the Table was determined from at least 3 separate experiments. h,,, gives the observed wavelengths of maximum absorption; E is the extinotion coefficient per mole of haem at the wevelength of maximum absorption. The values of E for the deoxy form (Q) were taken from Antonini & Brunori (unpublished results) for normal haemoglobin and from Antonini et al. (1964) for meso- and deutero- reconstituted haemoglobins; the values of e for the proteinsliganded with isocyanide (E& were determined from the experimentally observed ratio of ( exc/ea).

This shows that the dependence of Cllz on the molecular dimensions of the ligand is somewhat complex. In the case of three ligands (EtNC, Pr’NC and ButNC) which have the same “maximum length” (-7.7 A), but different bulkiness, there is a significant decrease in afllnity with increase in bulkiness, as pointed out originally by St George & Pauling (1951). In the case of ligands which have the same mass but TABLE

2

Apparent equilibrium constant (I = (C,,,)-‘) and Hill parameter (n) for the reaction of human haemoglobin with isocyanides as determined from direct binding experiments in O-2 M-phosphate, pH 7.0, 20X’, low5 M-huemoglobin Ligand

I x 10-a @-1)

MeNC EtNC Pr’NC Pr”NC ButNC Bu’NC

3.5 50.4 9.0 50.3 3.6 kO.9 7.3 11.8 0~11*0~08 -

?a 23+0.1 2.4&0.1 2.2f0.2 2.3&0.4 l-S&-0.4 -

I x 10-s @-‘I

13 -w 3.3 (&) 0.0&a, 4.0 (b)

(a) Values at pH 66, 0.13 M-phosphate and 25”C, from data of St George & Pauling (1961). (b) Value at pH 7.0, 0.1 M-phosphate and 22”C, from data of Olson t Gibson (1970). Cllz is the free ligand concentration which corresponds to a fractional saturcttion, E = 0.5. Each entry in the Table gives the average value with 95% confidence limits as determined from at least 5 independent experiments.

426

M. BRUNORI

ET AL.

differ in shape (Pr”NC versus Pr’NC or Bu”NC versus ButNC), it is observed that the affinity of the more extended form is greater. This finding suggests that steric hindrance effects represent more serious handicaps ta the binding as the bulkiness of the ligand increases in respect to its total length. The value of n for four isocyanides, MeNC, EtNC, Pr’NC and Pr”NC, is about 2.2 to 2.4, which is considerably lower than that for 0, (n=2*8); in the case of ButNC it is lower still, namely 1.8. This is discussed below. (c) The reaction with nwmd hae~globin

as measured by partition

In the case of a simple system (n=l) it follows from the mass law that the partition coefficient between any two ligands (such as OS and any isocyanide, NC) is given by:

Q = --=PeW KM [Fe%1 WI

1 K

(1)

where I and K denote the affinity constants for the binding of NC and 0,, respectively, to the haemprotein. Thus the value of Q should be independent of the ratio ([O,]/[NC]). This seems to be true for the partition between OS and isocyanides in the case of the isolated (Yand fl chains (see Talbot et al., 1971). The situation is, of course, more complex in the case of haemoglobin, for which n#l. However, if the shape of the binding curve for two ligltnds is the same, so that the two equilibrium curves can be superimposed by introduction of an appropriate scale-factor, then the partition coefficient should also be constant for all values of the ratio ([O,]/[X]) (Wyman, 1948,1904). Thus when the second ligand is carbon monoxide the partition coe&ient appears to be essentially independent of the relative concentration of OS and CO, as expected from the identity of shape of the ligand equilibrium curves (Allen & Root, 1957; Joels & Pugh, 1958; Wyman, 1964; Bonaventura & Riggs, 1968; Antonini & Brunori, 1970). The same constancy of Q would not be expected to hold in the case of OS (observed n=2*8) and the isooyyanides (observed n=2*2 to 2*4)-t. A total of 23 partition experiments were performed, with haemoglobin at 5 x 10m5M or 5 x 10m6 M, and MeNC, EtNC or Pr’NC in 0.2 M-phosphate, pH 7.0, or 0.05 Mborate, pH 9-l. In all cases the value of Q decreased uniformly with decreasing values of [O,]/[NC]; examples rare given in Table 3. The change in the value of Q with [O&NC] is discussed below. For the three isocyanides tested, the value of QI,.p=Q, corresponding to the point where [FeNC] = [FeO,], agreed well with the ratio of ~~~~~~~~~obtained from the binding curves in the presence of dithionite. For each of the three isocyanides, Ql,a was independent of pH (i.e. the same in O-2Mphosphate, pH 7.0, or 0.05 M-borate, pH 9.1) and of protein concentration (i.e. the same at 5x10m6 M or 5~10~~ M). (d) Efsects of different variables on the equilibrium The Bohr effect for MeNC, EtNC and Pr’NC binding to haemoglobin is shown in Figure 1 in terms of log Cl,= as & function of PH. The similarity of the curves for the various isocyanides with those for 0, and CO confirms the earlier conclusion that the t According to Roughton (1970) the partition coefficient between O2 end CO is also dependent on the relative concentration of the two ligande, ELfaot which may be due to a phenomenon similar in type, although smaller in magnitude, to that clearly demonstrated here for the o&se of the partition between O2 and isocyanides (see Jeo Disoussion).

REACTION

OF

ISOCYANIDES TABLE

WITH

HAEMPROTEINS

427

3

Data for the partG3n between oxygen ad kocyanidee for noma.! hum&n huemoglobin at 20°C Ligend

MeNC

WI

(h&ems/l.)

PH

5x10-6

9.1 (0.06 Mborate)

EtNC

6~10-~

(042. phosphete)

PrlNC

6x10-B

(0:: Mborate)

[NC] x 10s (mOl+l.)

[HbNC] [HbOa] + [WNC]

I 6

Q

0.96 1.91 3.09 6.42 8*86 14.2

0.19 0.29 0.39 0.60 0.63 0.72

0.28 0.14 0.087 0*060 0.030 0*019

0.066 0.058 0.066 0.060 0.061 0.046

0.42 0.84 1.67 2.90 6.32

0.37 0.62 0.67 0.77 0.86

0.64 0.32 0.16 0.093 0.061

0.37 0.34 0.32 0.30 0.30

0.47 1.03 1.96 3.32 ii.94

0.18 0.30 0.46 0.67 O-69

0.68 0.26 0.14 0.081 0.046

0.13 0.11 0.11 0.10 0.10

All experiments were performed adding suooessive samples of isocyanide solution kept in equilibrium with sir ([O,] = 2.7 x 10-4 M).

O5

(02) (NC)

I 8

I 7

to a haemoglobin

I 9

PH

Fro. 1. Bohr effeot for &NC (6lled symbols), EtNC (half-filled symbols) and Pr*NC (open symbols) binding to haemoglobin at 20°C. Ciroles refer to 0.2 r+r-potassium phosphate, triengles to 0.06 ~-sodium borate end squeres to 0.4 ar-sodium acetate buffer. The continuous line refers to the 0s Bohr effect determined under the seme conditions. Note: log Cl/s for EtNC is shifted above by 0.6.

428

ET AL.

M. BRUNORI

linkage between proton binding and binding of ligands to the haem iron is independent of the nature of the ligand (Wyman, 1964; Antonini & Brunori, 1971). The shape of the binding curves for MeNC and Pr’NC depends somewhat on pH ; the value of n is roughly constant (2.2 to 2.6) between pH 6 and 8.5 and decreases at higher and lower values, much as reported earlier for EtNC (Anderson, Antonini, Brunori & Wyman, 1970). Likewise, the effect of protein concentration and of ionic strength on the shape of the ligand binding curve is in general agreement with the findings reported earlier for EtNC. (e) Isocyanide binding by modi$ed hoewwglobins Table 4 reports the apparent equilibrium constants and the values of n for the reaction of MeNC, EtNC and Pr’NC with normal (proto-) haemoglobin, and with reconstituted meso- and deutero-haemoglobins. It will be seen that the substitution of proto-haem by either meso- or deutero-haem does not alter the relative affinities for the various ligands. Thus, in all cases, O,>EtNC>(MeNC, Pr*NC) ; also for all three haems, the value of n is consistently lower for the isocyanides than for 0,. Table 5 gives similar data for the reaction of Pr’NC with several modified haemoglobins. It shows that any chemical modification studied altered the behaviour toward the two ligands, Pr’NC and 0,, in a similar fashion. Thus in cases (e.g. iodoacetamide-treated Hb) in which the co-operativity is largely maintained in the 0, binding, the same is true for Pr’NC binding; likewise, chemical modifications (e.g. carboxypeptidase A-treated Hb and Hb Rainier) which totally abolish cooperativity in 0, binding, also abolish co-operativity in the binding of Pr’NC. Moreover, chemical modifications which greatly increase 0, affinity, generally greatly increase Pr’NC af%lnity.

TABLE

4

Apparent equilibrium constant (I = (C,,,)-‘) and Hill parameter (n) for human hemoglobin and reconstituted haemoglobins as determined from ligand binding curves in O-2 M-phosphate, pH Y-0, 20°C

Ligand

Normal Ix 10-a (M-l)

02

8%(4

MeNC EtNC PriNC

3.QO.4 9.010.3 3*6& 0.9

haemoglobin 12

28 (a) 2.3 2.4 2.2

Meso-reconstituted haemoglobin Ix 10-a It (M-l)

276 (b) 18.9k3.6 28.6f9.5 14.3k2.6

1.6 (b) 1.1 1.6 1.1

Deutero-reconstituted heemoglobin Ix 10-S 12 (M-l)

100 (c) 7.611.1 27.4k9.8 14.4+3.0

1.6 (c) 1.0 1.0 1.0

(a) From Antonini & Brunori (1971). (b) Values at 0.1 M-phosphate, pH 7.2, and 3O”C, from data of Rossi Fanelli, Antonini & Caputo (1969). (c) Values at 0.1 r,r-Tris, pH 7.0, and 3O”C, from data of Rossi Fanelli & Antonini (1959). All other data in the Table are the author’s, given as the average value with 95% confidence limits as determined from et least 3 independent experiments.

REACTION

OF

ISOCYANIDES

WITH

429

HAEMPROTEINS

TABLE 5

Apparent equilibrium constant (I = (C,,,)-*) and Hill parameter (n) for the binding of oxygen and isopropyl tiocyanide to human haemoglobin ancl moo%$edhuemoglobins as determined from ligand biding curves in 0.2 M-phosphate, pH 7-0, 20°C Oxygen Ix

10-3

Pr’NC 12

(ma-‘) Normal Hb IAAt Hb PCMB Hb CPA Hb Hb Rainier 8- sH ahains c~-~s chains

56 120 610 1700 948 1400 1200

64 (b) (c) (d) (e) (f) (f)

n

1x10-3

W’) 2.8 2.8 2.6 1.0 1.0 1.0 1.0

(a) (b) (c) (d) (e) (f) (f)

36f0.9 12&2 2.9*@8 18’7f62 156f74 180f70 23Ok 60

2.2 2.0 1.6 1.0 1-l 0.9 0.96

For authors’ data in the Table, each entry is the average, with 95% confidence limits as determined from at least 3 independent experiments. (a) From Antonini & Brunori (1971). (b) From Taylor et al. (1966). (c) From Giardina et al. (1971). (d) From Antonini et al. (1961). (e) From Amiconi et al. (1972). (f) Values in 0.15 Mphosphate, pH 7.6, 20°C. t Abbreviations used: IAA, iodoacetamide; PCMB, p-ohloromerouribenzoate; CPA, carboxy. peptidase A.

(f) Isocyanide binding to artiificial CN-met intermediates (a +cN /3 and ap +cN) Since the CN-met intermediates are unstable in the presence of dithionite, determination of the atkity constant of theae molecules for the various isocyanides was achieved by measuring the partition coefficient (Q) with 0,. Since n=l for 0, binding, it is assumed that n=l also for isocyanide binding; therefore Q should remain constant as ([O,]/[NC]) varies, and therefore be calculable as discussed above (see also Talbot et al., 1971). Values of Q for the two CN-met intermediates are reported in Table 6 together with similar values for the two chains and for haemoglobin. In a general way, the partition coeflkient of the ferrous chains in each intermediate resembles that of the corresponding free chains, this effect being particularly evident for the binding of TABLE 6

Partition we@cient (Q) between oxygen and isocyanides for human haemoglobin, 01and i3 chains, and arti$.cial CN-met intermediates in 0.2 M-phosphate, pH 7.0, 2O”C, protein concentration 5 x 10T6 M

a +0ti 4 B a+CN B Hb

MeNC

EtNC

Pr’NC

0*16~0~02 0.14*0*04 0.08f 0.02 0~06~0~01 0*06&0.03

0.83&0*17 0*73&0.18 0.34*0*10 0*33&0*08 0.32&0.07

0.24&0*03 0.14*0.03 0*13&0.02 0*11*0*02 0~10~0~01

Values given in the Table are the average values with 96% confidence limits, each entry based on at least 4 independent experiments involving at least 2 different preparations. 28

being

430

M.

BRUNORI

ET

AL.

MeNC, and of EtNC. For both intermediates, the partition coefficient between EtNC and 0, is independent of pH (from 6 to gel), showing that the alkaline Bohr effect for EtNC is very similar to that for 0, (Brunori et al., 1970). By using the values of Q from Table 6, and previous data for the apparent equilibrium constants with oxygen (K), we have calculated the apparent equilibrium constants with the isooyanides (I), which are given in Table 7. It is seen that for all three isocyanides as well as for O,, the order of afllnities is maintained: free chains > CN-met intermediates > haemoglobin. TABLE

7

Apparent equilibrium constant (I) for the reaction of haemproteins with isocyanides (at pH 7.0 and 25°C) as calculated from their direct binding with oxygen? and partition$ between 0, and NC KxlO-5

a 4 +CN B a+CN B Hb

Cahu18ted,IX10-6

02

MeNC

EtNC

12t 3.6t 14t 3.67 046t

1.8 (2.1)s OxFiO$ 1.1 (2.3)s 0*22$ 0.034 (0.035)5

10 (13)§ 2.61 4.8 (7.9)s 1*2$ 0.18 (0.09)§

Pr’NC 2.9 (2*3)§ 0.50$ 1.8 (l.S)§ 0.40$ 0.066 (O*OSS)§

t K for direct binding with oxygen. Data from Bnmori, Noble, Antonini & Wyman (1966) and Brunori et al. (1970). $ Partition data taken from Table 6. 5 Values in parenthesis, given for comparison, were det,ermined from direct binding of isocyanide (data from Table 4 and from Talbot et al., 1971).

4. Discussion The present work confirms and extends earlier results which indicated that any chemical modification of haemoglobin which raises the ai%nity for one ligand also raises it for other ligands. This is shown in Table 8 in which a comparison of the binding constant for different ligands relative to that for oxygen is given for many of the available cases. Likewise, chemical modifications which reduce co-operativity for one ligand, do so for all (Antonini & Brunori, 1971). These findings allow one to set, on a very general basis, the conclusion already reached comparing 0, and CO, that in its most general aspects the behaviour of haemoglobin is independent of the nature of the ligand, although in detail some of the functional properties may differ from one case to another. For normal haemoglobin in phosphate buffer, the value of n in the Hill equation is uniformly lower for the isocyanides (2.4 to 2.2) than for 0, or CO under comparable conditions (~2.8). At this stage it is difficult to decide unequivocally whether the decrease in the value of n is at all determined by a drop in the over-all free energy of interaction. A very likely interpretation, however, which finds support in parallel kinetic data (Olson & Gibson, 1970) and in analogies with the Redox equilibrium in haemoglobin (Antonini & Bnmori, 1970), may be given in terms of intrinsic functional heterogeneity between the OLand @chains, which manifests itself in a decrease in the value of n, even if the over-all interaction energy remains approximately constant. It seems reasonable that the chains, which are essentially alike with respect to the

Hb Meso Hb (R) Deutero Hb (It) Mb Meso Mb (R) Deutero Mb (R) &-am § as* 4’“” PC”” SH Is a+CN B (lOOH (low loot loot lOO$ loot loot lOO$

(lOOH

lOOi loot lOO$

l6S {‘3*1)$

111 173 {14If 9.5%

24 x 103t 25 x 103t 23 x 103t 34 x 103-f -

[51 xYo”,t -

6.3$ 6.81 7.6x -

MeNC

36 x 103t -

co

563 {33X

2S#

108$ C731$

(1W my

272 wvt

lot

162

EtNC

KxlKo~

{:? 13f {11X

1 13x 1w

(1%

14$

6.5$ 5.2$

Pr ‘NC

of the a@nity of several huemproteins for various ligands (K,) relative to that for oxygen (K,,), Conditions: pH 7.0, phosphate buffer, 20°C

8

(R), Reconstituted; ( ), sperm whale Mb; [ 1, horse Mb; { }, values from the partition coefficients; t, data from Antonini Tables 2, 4, 6 and 7, and from Talbot et al. (1971). SAbbreviation used: PCMB, p-chloromercuribenzoate.

Comparison

TABLE

& Brunori

0.76$ 0.53$ -

0.19$ 0.221

6,t

0*20$

ButNC

(1971);

which is arbitrarily

$, data from

set af 100.

M. BRUNORI

432

E!l’

AL.

smaller ligands, (0, and CO), become progressively different with respect to the larger and more bulky isocyanides, the heterogeneity being either intrinsic or linked to the binding of a third component to a particular chain. For haemoglobin, the observed decrease of Q with decreasing [O,]/[NC] might plausibly be rationalized on the basis of an heterogeneity between the two types of chains for the larger ligands. Then for the two chains:

[HbaNCl

PRO,1

[Hb,PCl

= P’Q, and fHbsO,l = p’Q8

where p’ = [NC]/[O,J, end Qi#Q@. From this

(2)

where r = Q,/Qp,and P” = (Q, Q# WJJI/P~. As the concentration of isocyanide increases from zero, p” rises; at the point where [HbNC] = [HbO,], p” = 1 and z = 1. At this point the experimentally determined over-all value of Q equals (QaQB)*.Call this point Q+ and the corresponding value of the isocyanide concentration [NC,,,]. The Q measured at any other point (QJ should then be related to Ql,s by the equation

-=Q, Q1/2

(r + 1) + Wt WGl/W,l) (r + 1)(Wtl/lN~1,2l)

+2(r)+

(3)

The results show considerable scatter but the best over-all fit of the equation to the experimental data indicates that one type of chain in the assembled haemoglobin molecule has an &lnity for isocyanide which is three to four times higher than the other, which is not unreasonable. Although from these results it is impossible to establish which of the two chains displays the highest atiity for the ligand, a reasonable guess, on the basis of kinetic results with isolated chains (Talbot et al., 1971) and with haemoglobin (Olson & Gibson, 1970), is that even in the assembled molecule the /l chains have, on the average, sn affinity higher than the corresponding 01chains. The variation of the over-all free energy of binding of the various isocyanides to haemoglobin or to the isolated Hb chains is well established; the trend in &inity is shown by the results reported in Table 2 for the case of Hb, and by the data in Table 4 of a previous paper (Telbot et al., 1971) for the isolated 01and fi chains. It is obvious from the available d&a that, independently of the presence or absence of co-operative phenomena in the ligand binding process, the afllnity is maximal for EtNC and minimal for ButNC. A reasonable explanation of the observed trend in binding constants can be provided on the basis of the knowledge acquired from inspection of the three-dimensional atomic model of haemoglobin (due to Dr M. F. Perutz), that the aliphetic carbonchain is preferentially packed towards the interior of the molecule, in a region which is very rich in non-polar side chains. Therefore the binding of the alkylisocyanides to haemoglobin should be favoured by the gain in free energy due to the transfer

REACTION

OF

ISOCYANIDES

WITH

HAEMPROTEINS

433

of the hydrocarbon side-chain of the ligand from water to the non-polar environment provided by the protein; this energy term should become more favourable for binding as the length of the side-chain becomes larger and larger. In principle this energy term could be estimated quantitatively from measurements of the solubility of the alkylisocyanides in water. However, as the ligand side-chain becomes larger, packing in the pocket provided by the protein, which presumably can be obtained only at the expense of a certain degree of rearrangement in the amino-acid side-chains, becomes more and more difficult due to steric hindrance effects. In fact, fitting the ligands in the proper position in the pocket of the a and j3 chains results in a number of short contacts which, for n-butyl isocyanide and tertiary-butyl isocyanide, are given in Table 9 (date of Dr M. F. Perutz). Since ethyl isooyanide displays, for all haemproteins, the highest afEnity, it appears, on the basis of these simple considerations, that in this case the best compromise between unfavourable sterio hindrance effects and favourable free energy of transfer to the protein’s non-polar pocket is achieved. TABLE 9

Short contact8 between the a and tY3chains of haemoglobin and two alkylisocyanides n-Butyl isocyanide a chainfl

j3 chains

Leu BlO(29)

val Ell(67) Ala BD(27) Leu G8( 106)

Leu Val Leu Ala Gly

GS(lO1) Ell(62) G12( 105) BD(28) B6(26)

Leu E12(68)

Tertiary-butyl

isocyanidef

a chains

fl chains

Val Ell(62) Leu BlO(29) Leu GS(101)

val Ell(67) Leu BlO(28) Leu GS(106)

t The distanoes between these groups and the tertiary-butyl the same.

groups in o(and fi are not necessarily

It is a pleasure to express our appreciation to Dr M. F. Perutz for discussions and invaluable information concerning the mode of binding of alkylisooyanides in haemoglobin as well as for providing the values for short contacts given in Table 9. We thank Dr K. H. Winterhalter for kindly giving a sample of purified Hb Rainier and Mr A. Pescarollo for his exoellent technical assistance. A grant from the National Science Foundation to one of us (J. W.) is gratefully acknowledged. REFERENCES Allen, T. A. & Root, W. S. (1957). J. AppZ. PhyeioZ. 10, 186. Amiconi, G., Winterhalter, K. H., Antonini, E. & Brunori, M. (1972). FEBS Letters. In the press. Anderson, N. M., Antonini, E., Brunori, M. & Wyman, J. (1970). J. Mol. BioZ. 47, 205. Antonini, E. & Brunori, M. (1970). Ann. Rev. Biochem. 39, 977. Antonini, E. t Bnmori, M. (1971). Hemoglobin and Myogbbin in their Reaction-s with Garuk. Amsterdam : North-Holland Publishing Company.

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