Immunology, 1971, 20, 649.

Complement Fixation by the F(ab')2-Fragment of Pepsin-Treated Rabbit Antibody

K. B. M. REID

Department of Biochemistry, University of Oxford, South Parks Road, Oxford

(Received 7th August 1970)

Summary. The F(ab') 2-fragments of rabbit anti-ovalbumin and anti-humanserum albumin IgG fixed between 30 and 50 per cent of guinea-pig complementwhen they were aggregated with the appropriate antigen. The fixation took placeat 370 but not at 40 and was completely inhibited by 0-005 M EDTA. The fixationwas more efficient when preformed immune aggregates were used rather than allow-ing the aggregates to form in the fixation mixture. The loss in haemolytic activitywas probably due to the fixation of one or more of the late components (C3 to C9)ofguinea-pig complement since nosignificantfixation ofthe Cl and C2 componentscould be found. These results may help to explain previous conflicting reports onthe ability of 5S antibody to fix complement. The results also suggest that rabbitantibody F(ab')2 may fix complement by a pathway which utilizes only the com-ponents C3 to C9 thus bypassing the more usual pathway initiated by C l activation.

INTRODUCTION

There have been conflicting reports about the ability of the F(ab') 2-fragment ofpepsin-treated rabbit antibody to fix guinea-pig complement. Several authors (Tarantaand Franklin, 1961; Ishizaka, Ishizaka and Sugahara, 1962; Ovary and Taranta, 1963)could not detect any complement fixation by antigen-F(ab')2 aggregates, whereas others(Amiraian and Leikhim, 1961; Schur and Becker, 1963; Chan, Jaquet and Cebra, 1964;Jaquet and Cebra, 1965; Isliker, Jacot-Guillarmod, Waldesbtihl, von Fellenberg andCerottini, 1968) did detect some. The maximum fixation of complement by the antigen-F(ab') 2 aggregates was reported to vary between 30 and 50 per cent of the total comple-ment added. It would appear that only a certain portion of guinea-pig complement isavailable for fixation by antigen-F(ab')2 aggregates. It was decided to re-examine thisportion of complement which was fixed by antigen-F(ab') 2 aggregates in order to: try todetermine why conflicting results had been obtained previously; establish whether or notCl was fixed by F(ab')2 since it has been reported that the Clq binding site is located onthe Fc region of the IgG molecule (Isliker et al., 1968).

MATERIALS

Pepsin (2 times crystallized, batch 69B-2000) was obtained from Sigma (London)Chemical Co., London, S.W.6; carboxymethyl-cellulose (CM-cellulose) and diethyl-aminoethyl-cellulose (DEAE-cellulose) from H. Reeve Angel and Co., London; SephadexG-150 from Pharmacia Fine Chemicals, Uppsala, Sweden; sheep erythrocytes fromWellcome Reagents Ltd, Wellcome Research Laboratories, Beckenham, England.

649

Ovalbumin (HEA) was recrystallized three times after isolation from hen egg white(Kekwick and Cannon, 1936). Human serum albumin (HSA) was a gift from the ListerInstitute Blood Products Unit.

METHODSPreparation of anti-sera

Anti-ovalbumin (anti-HEA) and anti-human serum albumin (anti-HSA) were pre-pared by injection of alum-precipitated ovalbumin and human serum albumin intorabbits as described by Porter (1955).

Isolation of anti-HEA and anti-HSA IgGRabbit anti-HEA and anti-HSA IgGs were isolated by precipitation from the immune

sera with Na2SO4 (Kekwick, 1940), purified on DEAE-cellulose and finally freeze-dried.

Preparation ofF(ab') 2: pepsin digestionThe digestion of anti-HEA and antiP.HSA IgG with pepsin was performed as described

by Jaquet and Cebra (1965). The IgG preparation (300 mg) was dissolved in 100 mmsodium acetate buffer, pH 4-5 (20 ml). Crystalline pepsin (6 mg) was added and themixture incubated at 370 for 18 hours. After 18 hours a small amount of precipitate wasremoved by centrifugation, the pH raised to 8 with 1 N NaOH and Na2SO4 (25 g/l00 ml)added, dropwise, at 250, with stirring to a final concentration of 19 g/l00 ml. The heavyprecipitate which formed was separated by centrifugation at 200 and dissolved in 20 mmTris (hydroxymethyl) methylamine (Tris) buffer, pH 8-0 (5 ml).

Sephadex gel-filtrationThe F(ab')2 preparation was fractionated on Sephadex G-150 at room temperature.

The sample in 0.02 M Tris buffer (5-7 ml) was applied to a Sephadex G-150 column(2-5 x 95 cm) equilibrated with 0-02 M Tris buffer, pH 8-0. The column was run, byupward flow, at a rate of 10 ml/hour and fractions of 4 ml were collected. The effluent wasmonitored at 280 my. One major and two minor peaks were obtained. Only the majorpeak was pooled, re-fractionated on Sephadex G-150, and characterized.

Preformed immune aggregatesF(ab')2 preparation and whole IgG were incubated with antigen, at the equivalence

point, for 2 hours at 370 and then at 40 for 16 hours. The precipitates were washed twice in0-02 M Tris buffer, pH 8f0, and finally resuspended in Tris complement buffer.

Protein estimationProtein was estimated by the method ofLowry (Lowry, Rosebrough, Farr and Randall,

1951). The moles of IgG and F(ab')2 present in each sample of immune aggregates wasderived from the antibody protein content of each sample assuming a molecular weight of150,000 for IgG and 90,700 for the F(ab')2 fragment (Jaquet and Cebra, 1965).

Buffer used in complement studiesTris complement buffer (TCB; 50 mm Tris, 40 mm HCl, 0-15 mm CaCl2, 0-5 mm

MgCl2, 98 mm NaCl, pH 7.5) was used in place of Veronal-buffered saline for haemolysin

650 K. B. M. Reid

Complement Fixation by F(ab') 2-Fragmentand complement titrations. For storage of C 1 and C2 preparations and all reactionsrequiring low ionic strength-isoosmotic conditions, Tris complement buffer with sucrose(TCB-S) was used (50 mm Tris, 40 mm HCI, 0 15 mm CaCl2, 0 5 mM MgCl2, 25 mMNaCl, 146 mm sucrose, pH 7 5). Complement ethylenediamine-tetraacetate (C-EDTA)was prepared by appropriate dilutions of whole complement with ice-cold Tris comple-ment buffer-EDTA (TCB-EDTA) which was free of Ca+2 and Mg+2 and contained50 mm Tris, 40 mm HCl, 98 mm NaCl and 10 mm EDTA and had a pH of 7-5.

Preparation of haemolysin and complementRabbit haemolysin was prepared using sheep erythrocyte stromata as described by

Mayer (1961). The immune sera were heated at 560 for 30 minutes before dispensing in1-0 ml aliquots and freezing at -20°. Whole complement was prepared by bleedingnormal Hartley-strain guinea-pigs by cardiac puncture and allowing the blood to clot at40 for 2 hours. The pooled sera were either frozen at -700, in 1P0 ml aliquots, withoutfurther treatment, to be used as a source of whole complement or were processed imme-diately in preparing functionally pure C1 and C2.

Preparation ofpartially purified complement componentsCl was prepared from whole guinea-pig serum as described by Linscott (1968), except

that the gel filtration step was missed out. The final precipitate was dissolved in TCB-S,clarified by ultracentrifugation, and the supernatant dispensed in 10 ml aliquots andstored at -70°.C2 was prepared according to Nelson, Jensen, Gigli and Tamura (1966) by CM-

cellulose chromatography of the 3 0 M (NH4)2SO4 precipitate. The preparation wasadjusted to pH 6-0 before dispensing in I 0 ml aliquots and storage at - 700. Dilutions ofC2 were made in TCB-S.

Complement fixationPreformed immune aggregates in TCB, or alternatively antibody and antigen, were

incubated with undiluted guinea-pig serum (1.0 ml), at 370 for 60 minutes. (In initialexperiments fixation was performed at 40 for 18 hours.) Ice-cold TCB was added at the endof the fixation period and the tubes were centrifuged at 5000 g for 30 minutes.

In two experiments the fixation was performed in low ionic strength buffer, containingapproximately 100 CIH50 units, exactly as described by Ishizaka, Ishizaka, Borsos andRapp (1966). However, usually complement fixation was performed in an isotonic mediumplus undiluted guinea-pig serum, as described above, so that Cl, C2 and C-EDTAassays could be conveniently performed on the same sample.

Titration qf complement and complement componentsThe supernatants ofthe fixation mixtures were titrated for residual haemolytic activity as

described by Mayer (1961). Cl activity was determined as described by Ishizaka et al.(1966) using EAC4 prepared as described by Borsos and Cooper (1961). In the laterexperiments EAC4 was prepared according to the method of Borsos and Rapp (1967). C2titrations were performed according to the method of Borsos, Rapp and Mayer (1961)except that TCB-S was used during the interaction of EAC14 and C2 (Rapp and Borsos,1963). The late components (C-EDTA) were estimated as described by Mayer (1961)using EAC142 prepared with functionally pure C2 and EAC14 (Mayer, 1961).

651

Ultracentrifuge analysisPreparations were examined at a concentration of 4 mg/ml in 200 mm Tris buffer,

pH 8f0, in a Spinco Model E analytical ultracentrifuge at 200 and at 59,780 rev/min.

RESULTS

Purity of the F(ab')2 fragmentsThe purified F(ab')2 fragments of anti-HEA and anti-HSA IgG ran as single, sym-

metrical, peaks when re-run on Sephadex G-150. They also ran as single components inthe ultracentrifuge with an S020,w of 4-7. They showed no reaction in double diffusionwith goat anti-rabbit Fc but gave a strong reaction with goat anti-rabbit Fab.

100 _

75

X~~~~~

V

0~~~~

a) / 0

EI'50 --E0

25

-10 0 1I0 20 33log n mole F(ab') or IgG

FIG. 1. The percentage of complement fixed is plotted against the logarithm of the antibody protein (inthe form of preformed immune aggregates) present in the fixation mixture: *, anti-HEA IgG 60minutes at 370 or 18 hours at 40; o, anti-HEA F(ab')2 60 minutes at 370; A, anti-HSA F(ab')2 60minutes at 370; A, anti-HEA F(ab')2 18 hours at 4°.

Fixation of complementAs can be seen in Fig. 1, both anti-HEA and anti-HSA F(ab')2 showed complement

fixation, at 370, when aggregated with the appropriate antigen. This fixation was moreefficient when preformed aggregates, rather than aggregates formed by allowing antigenand F(ab')2 to interact during the fixation period, were used (Fig. 2). The preformedimmune aggregates were of the order of twenty times more efficient at the point ofmaximal complement fixation. The difference in efficiency was greater at lower fixationvalues (Fig. 2).

652 K. B. M. Reid

Complement Fixation by F(ab') 2-Fragment

The fixation by F(ab')2 aggregates was very dependent on the temperature used. As canbe seen, in Fig. 1, 0-23 nmole of aggregated anti-HEA F(ab')2 fixed 23 per cent of thecomplement in 60 minutes at 370 whereas 100 times that amount showed no fixation at allafter 18 hours at 4°. The same dependence on temperature was shown for preformedimmune aggregates of anti-HSA F(ab') 2. On the other hand, preformed immune aggre-gates of intact anti-HEA and anti-HSA IgG showed the same degree of complementfixation at 40 as they did at 37°. Anti-HSA IgG and anti-HEA IgG fixed complement in al-most an identical manner; for the sake of clarity, only fixation by anti-HEA IgG is shown inFig. 1.

40

30-

a)

a),E20

10-~~~~~

0~

0-10 0 0

log n mole F (ab')2

FIG. 2. The percentage of complement fixed in 60 minutes at 37° is plotted against the logarithm of theantibodyprotein present in the fixation mixtures:0, preformed immune aggregates ofanti-HEA F(ab')2;c, aggregates ofHEA and anti-HEA F(ab')2 allowed to form in the fixation mixture.

Although both the anti-HEA and anti-HSA F(ab') 2 aggregates fixed appreciableamounts of complement there was a definite limit to the amount they could fix. Theanti-HEA F(ab')2 aggregates fixed up to a maximum of 49 per cent of the complementand no more (Fig. 1). The same was found to be true for the anti-HSA F(ab')2 aggregatesused, only in this case the maximum amount ofcomplement that could be fixed was 32per cent (Fig. 1). This limit in the amount of complement fixed may be a function of thedegree of pepsin digestion since a second preparation of anti-HEA F(ab')2, when aggre-gated, fixed only 32 per cent of the complement (Fig. 2). Relatively small amounts ofintact anti-HEA IgG readily fixed over 90 per cent of the complement present (Fig. 1).Despite the difference in the maximum amounts of complement fixed by the F(ab')2aggregates and by intact IgG aggregates the efficiency with which each fixed that portionof complement available to them was approximately the same (Fig. 1).

653

Anti-HEA IgG subjected to the entire digestion (incubation at pH 4-5 for 18 hours, butwithout addition ofpepsin) and purification procedures used to obtain F(ab') 2 was found tofix complement in exactly the same manner as anti-HEA which had not been subjected tothese procedures.

It was also observed that addition of aggregates of anti-HEA IgG to serum which hadbeen depleted of 49 per cent of its complement activity, by F(ab') 2 aggregates, causedcomplete loss of complement activity. This showed that the portion of complement notfixed by the F(ab') 2-aggregates was readily fixed by IgG aggregates.

TABLE 1FIXATION OF COMPLEMENT COMPONENTS BY PREFORMED IMMUNE AGGREGATES OF:(1) OVALBUMIN PLUS ANTI-OVALBUMIN IgG; (2) OVALBUMIN PLUS ANTI-OVALBUMIN

F(ab') 2

Per cent of haemolytic activity lost*Sample IgG or F(ab')2

(nmole) C C1 C2 C-EDTA

(1) Anti-ovalbumin 0-67 60 40 41 60IgG plus ovalbumin 2-01 89 58 59 N.E.

3-38 92 73 N.E.t 91(2) Anti-ovalbumin 0-58 32 4 N.E. 16F(ab')2 plus ovalbumin 1-20 41 N.E. 0 58

340 48 5 0 635-60 49 0 4 646-10 49 4 5 6816-80 49 6 0 68

* The CH50, CIH50, C2H50 and C-EDTA H50 titres of 1 0 ml of undilutedguinea-pig serum were always in the range 150-170, 18,000-20,000, 16,000-21,000 and 170-250 respectively. Variation between duplicate control tubes didnot exceed 4 per cent in each experiment.

t N.E., not estimated.

Fixation qf complement componentsIn the C 1 fixation experiments performed in low ionic strength buffer, as described by

Ishizaka et al. (1966), the CIH50 titres of 10 ml aliquots of diluted guinea-pig serum,after incubation with 6-1 nmole of anti-HEA F(ab')2 aggregates, 3-4 nmole of intactanti-HEA IgG aggregates and buffer were 102, nil and 106 respectively. When repeated, induplicate, values of 98, nil and 101 were obtained. Therefore, there is no significantfixation of Cl by F(ab')2 aggregates in low ionic strength buffer.

It can be seen (Table 1) that extensive fixation of C1, C2 and C-EDTA was observedwith small amounts of intact anti-HEA IgG aggregates. On the other hand, only back-ground values for the fixation ofC 1 and C2 were obtained using large amounts of F(ab') 2aggregates whereas small amounts of the F(ab')2 aggregates fixed up to 68 per cent of thelate components (Table 1). As observed in the whole complement titration, only a certainpercentage of the late components appeared to be available for fixation by F(ab')2aggregates. Again, similar to the fixation of whole complement, the F(ab')2 aggregateswere almost as efficient as the intact anti-HEA IgG aggregates in fixing that portion ofthe late components which was available to them (Table 1).

Effect of 0 005 M EDTAWhen preformed immune F(ab')2 aggregates were incubated with guinea-pig com-

plement, and the whole fixation mixture made 0-005 M with respect to EDTA, no decrease

654 K. B. M. Reid

Complement Fixation by F(ab') 2-Fragment

in the activity of whole complement or the late components (C-EDTA) was observed inthe fixation mixtures. The fixation mixtures contained 7-1 nmole of F(ab')2 aggregateswhich was three times that required to fix 49 per cent of the whole complement and 63per cent of the late components when the fixation was performed in the absence of EDTA.

DISCUSSION

The difference in efficiency of complement fixation brought about by using preformedimmune aggregates of rabbit F(ab')2, rather than allowing aggregation to take placeduring the fixation period, was first demonstrated by Schur and Becker (1963). Theyfound that preformed immune aggregates of rabbit F(ab')2 were of the order of 30-100times more efficient in fixing complement than were the F(ab' ) 2 antibody-antigen aggre-gates formed in the presence of complement. Sandberg, Osler, Shin and Oliveira (1970)have pointed out that guinea-pig yl IgG, which was thought, originally, to possess nocomplement fixing activity, also displays this dependence on aggregation for efficientcomplement fixation. The rabbit anti-HEA F(ab')2 preparation used in this work wasdefinitely more efficient (about 20 times at the point of maximum fixation) at fixingcomplement when used in the form of preformed immune aggregates. The difference inefficiency was not as marked as has been reported for rabbit anti-HSA F(ab')2 aggre-gates (Schur and Becker, 1963) and guinea-pig yl anti-DNP-BGG IgG aggregates(Sandberg et al., 1970) but was of the same order as for a preparation of sheep anti-HSAF(ab')2 aggregates (Schur and Becker, 1963). It is well established that immune aggre-gates, prepared at equivalence, take a considerable period of time to effect maximumprecipitation of all the antigen-antibody complexes formed and it is also generally agreedthat antigen-antibody complexes become increasingly stable with time. Therefore oneexplanation of the difference in efficiency between the two types of aggregates in fixingcomplement perhaps may be that the F(ab')2 molecules in the antigen-antibody complexesundergo a slow conformational change during aggregation after their initial, presumablyfast, reaction with antigen and that this change in conformation enhances the fixation ofthe later components.The marked effect of temperature on the fixation of complement by F(ab') 2 aggregates

(Fig. 1) would appear to explain, partially at least, why several authors (Taranta andFranklin, 1961; Ishizaka et al., 1962; Ovary and Taranta, 1963) concluded that theF(ab')2 aggregates were completely inactive in complement fixation. These authors hadperformed their fixation assays at 40, a temperature at which F(ab')2 aggregates appearto be completely inactive in complement fixation. The other explanation, as pointed outby Schur and Becker (1963), may lie in the aggregation procedure used.The fact that fixation by F(ab')2 aggregates took place at 370 and not at 40 suggested

that the later components, principally C3, were involved in the fixation since C3 fixation iswell known to be dependent on temperature. Also, it has been reported that rabbitanti-HSA F(ab')2 aggregates do not bind Clq (Isliker et al., 1968). In the light of thesetwo facts it is perhaps not surprising that anti-HEA F(ab') 2 aggregates do not fix Cl andC2 (and presumably C4) but fix appreciable amounts of the later components (Table 1).Thus the F(ab')2 aggregates appear to lack the activation sites for Cl and therefore bypassthe Cl, C4 and C2 sequence and enter the haemolytic system via an attack on the latercomponents, possibly at C3. In this respect the behaviour of the F(ab')2 aggregates issimilar to that of guinea-pig yl IgG aggregates (Sandberg et al., 1970).

B IMMUN.

655

Since EDTA can completely inhibit the fixation of complement by the F(ab')2 aggre-gates (see also Schur and Becker, 1963) this suggests that they require a divalent cation toinitiate their attack on the late complement components despite their non-usage of C1and C2. Sandberg et al. (1970) found that EDTA blocked the cleavage of C3 by guinea-pig y1 IgG aggregates and suggested that either minimal activation of Cl took place orthat a site was produced by aggregation of the guinea-pig y1 IgG which activated aserum enzyme which could cleave C3 as the initial step in the utilization of the latercomponents. They favoured the second explanation since, like the F(ab')2 aggregates, theguinea-pig yl aggregates did not utilize any C2 during fixation.

There is some evidence that such a serum enzyme or factor (i.e. which would cleave, orinactivate, C3) does exist. Mareus, Shin and Mayer (1970) have extended the work ofGerwurz, Shin and Mergenhagen (1968) and have demonstrated that incubation ofendotoxic lipopolysaccharide with guinea-pig serum at 370 can produce a complex ofendotoxic lipopolysaccharide plus another factor which utilizes the later components ofcomplement preferentially and which cleaves C3 into fragments similar to those generatedby EAC4,2. More evidence for the existence of a serum factor involved in the cleavage ofC3 has been provided by studies on cobra venom factor (Muller-Eberhard, 1968). It hasbeen shown that purified cobra venom factor inactivated C3 in whole guinea-pig serum buthad no effect on purified C3. The inactivation of C3, by cleavage into fragments, requiredthe interaction of a fl-globulin of the serum with the purified cobra venom factor and thisinteraction was blocked by EDTA. In a wider study of about thirty anti-complementaryvenom factors Birdsey, Linderfer, From, Day, Pickering, Moberg and Gewurz (1970)found that they fell into two general groups; one, the cobra venom factor type whichformed a stable C3 fixing intermediate and consumed predominately the terminal com-ponents; the second, present in a variety of species, consisted of a factor which activated anenzyme which efficiently consumed earlier acting components. The preferential actionbetween C3 and a complex of zymosan and guinea-pig serum factor(s), first observed byPillemar, Glum, Lepow, Todd and Wardlaw (1954), has also been well documented(see Lepow, 1965).These observations would suggest that there is an alternative pathway of complement

fixation utilizing the components C3 to C9 and bypassing the more usual pathway initiatedby Cl activation. The results reported here support the idea that F(ab')2 aggregatesperhaps utilize this alternative pathway when they fix complement.The limit in the amount of complement fixed by rabbit F(ab') 2 aggregates (30-50 per

cent with respect to whole complement Fig. I, II; 68 per cent with respect to C-EDTA,Table 1), has been observed often before. This limit in the amount of complement fixedis not dependent simply on the period offixation since it was found that samples ofguinea-pig complement incubated for 1, 1, 2 and 4 hours, with F(ab')2 aggregates, all showed47-51 per cent fixation with respect to appropriate control tubes. Guinea-pig yl IgGaggregates also may fix only 50 per cent of the whole complement and 56 per cent of theC-EDTA available as suggested by the results of Sandberg et al. (1970; Tables II andIV). This could mean that one of the components which is fixed by F(ab')2 aggregatesis heterogeneous in the sense that only a certain percentage of this component mayrecognize the sites produced by the aggregation of F(ab')2. In this connection it is ofinterest that Alper and Propp (1968) have reported that there are seven allotypes ofhuman C3. Another explanation for the inability of the F(ab')2 aggregates to fix all thecomplement available, could be that there is a limiting amount of a certain factor in the

656 K. B. M. Reid

Complement Fixation by F(ab') 2-Fragment 657

guinea-pig serum which, on interaction with F(ab')2 aggregates, fixes the later componentsselectively. To see if larger amounts of such a factor (giving a greater percentageof complement fixation) exist in human and rat sera the fixation experiments were re-peated using normal human and rat sera. The maximum fixation by the F(ab')2 aggre-gates, under conditions which gave 49 per cent fixation of guinea-pig complement, was56 per cent using human serum and 29 per cent using rat serum. Therefore if this hypo-thetical factor does exist it appears to be present in much the same quantities in the seraof three very different species.

Tentative evidence that there is a limiting amount of a factor in guinea-pig serum,which F(ab')2 aggregates require in the utilization of the later components, was obtainedby examining serum which had been treated with F(ab')2 aggregates, at 370, to givemaximal fixation (i.e. approximately 49 per cent; Table 1 and Fig. 1). The residualcomplement activity could be readily fixed by IgG aggregates, thus giving 100 per centtotal fixation. The residual activity could also be partially fixed (giving up to 73 per centtotal fixation) by F(ab')2 aggregates which had been incubated at 40 for 18 hours, or 170for 1 hour, with guinea-pig serum and then spun down and washed twice with ice coldbuffer prior to incubation with serum which had been treated at 370 with the F(ab')2aggregates. The residual activity was not, as expected, fixed to any significant extent byF(ab')2 aggregates which had been treated with serum at 370 for 1 hour. These obser-vations suggest that a complex may be formed at lower temperatures (4-170) betweenF(ab')2 aggregates and a serum factor and that this complex is active, and is utilized, infixing the later components of complement at 370.The results presented pose a new problem. If it is accepted that F(ab')2 aggregates

definitely do not fix C1, then either each rabbit IgG molecule has the ability, whenaggregated, to initiate attack on the complement system by two pathways; or, there aretwo populations of rabbit IgG molecule, one of which behaves like guinea-pig y1 IgG. Inthis context it would be of great interest to examine the F(ab') 2 aggregates of guinea-pigy 1 and v2 IgG to see if either, or both, showed the ability to fix the late components(C3 to C9) preferentially.

ACKNOWLEDGMENTSI would like to thank Professor R. R. Porter for his help and advice during the course of

this work and Dr E. L. Becker for his critical assessment of the manuscript. The work wascarried out during the tenure of an I.C.I. Research Fellowship.

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