Comparative immunochemistry of bacterial, algal and plant ferredoxins

Biochimica et Biophysica Acta, 490 (1977) 120-l 3 I © Elsevier/North-Holland Biomedical Press BBA 37548 COMPARATIVE IMMUNOCHEMISTRY PLANT FERREDOXINS

OF B A C T E R I A L ,

ALGAL AND

ELISHA TEL-OR% RICHARD CAMMACK ~, K. KR1SHNA RAO b, LYNDON J. ROGERS¢, WILLIAM D. P. STEWART a and DAVID O. HALLo ~Departnlent o/" Biological Sciences, Universit.v of Dundee, Dundee, bDepartment o! Plant Sciences, University o["London, King's College, London, and ¢Department of Biochemistry, University College of Wales, Aberystwyth ( U.K.) (Received May 20th, 1976) (Revised manuscript received September 9th, 1976)

SUMMARY 1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 [4Fe-4S] ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green alga Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris. The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins. 2. Antibodies to the bacterial ferredoxins reacted to a significant extent only with their homologous proteins. On the other hand, antibodies to the plant and algal ferredoxins showed cross-reaction with other ferredoxins. There was a correlation between the degrees of immunoprecipitation and the similarity in amino acid sequences. These results suggest that the method can be used as a marker in taxonomic studies. 3. The interaction of the antibodies with the five native ferredoxins was compared with the reactions with their apoproteins. In each case the degree of interaction was different. This behaviour was interpreted as due to an influence of tertiary structure on the antibody-antigen interaction.

INTRODUCTION Iron-sulphur proteins have a broad spectrum of function in the metabolism of carbon and of nitrogen and hydrogen, and in various bioenergetic pathways of higher plants, algae and bacteria [1 4]. Thus much effort has been invested recently ill obtaining a better understanding of their structure and function [5-6]. The possible use of immunological technique has been investigated on a restricted range of ferredoxins [7-11]. In this paper we present a detailed study of ferredoxin-antiferredoxin interrelations using antibodies to ferredoxins from five species of higher plants, algae and bacteria. These antibodies have been cross-reacted with ferredoxins from 30 organisms, with both 2Fe-2S centre and 4Fe-4S centre ferredoxins being represented.

121 The effectiveness of apoferredoxins in eliciting antibody responses has also been investigated. The overall results obtained are discussed in relation to the primary structure and conformation of the different ferredoxins, and in relation to possible phylogenetic affinities of the organisms from which the ferredoxins were obtained. MATERIALS AND METHODS

Ferredoxins Clostridium pasteurianum, Chromatium strain D, Bacillus stearothermophilus and Desulphovibrio vulgaris were obtained ti'om the Microbiological Research Establishment, Porton Down, Wilts. U.K. ; Chlorobium limicola from Dr. J. M. Olson, Brookhaven National Laboratory, N.Y., U.S.A. ; Clostridium acidi-urici from Dr. G. Heden, Karolinska Institute, Stockholm, Sweden; Scenedesmus obliquus and Euglena gracilis cells from Dr. C. J. Soeder, Coal Research Institute Dortmund, G.F.R. ; Cyanidium cells from Dr. H. Metzner, University of Tfibingen, G.F.R.; Mastigocladus cells from Dr. H. Zuber, Eidgenossische Technische Hochschule, Zurich, Switzerland; Spirulina platensis from Mlle. G. Clement, Institute Franqais du Petrole, Rueil-Malmaison, France; Medicago sativa (alfalfa) from Dr. D. Arckoll of Rothamsted Experimental Station, Herts. U.K.; Equisetum sp. from Dr. L. Packer, University of California, Berkeley, U.S.A.; Spirulina maxima from Ing. H. Durand-Chastel, Sosa Texcoco, Mexico, and Spinacia oleracea (spinach) from local markets. Ferredoxins were isolated from the above material according to the methods of Rao et al. [12] or Mullinger et al. [13] except for the ferredoxins from Nostoc strain MAC, Chlorogloea (Chlorogloeopsis) fritschii, Aphanocapsa 6714, Anabaena flos-aquae and Porphyridium cruentum which were isolated as described by Hutson and Rogers [14] and Andrew et al. [15]. Ferredoxin from Porphyra umbilicalis was isolated according to Andrew et al. [16]. The following kindly provided gifts of ferredoxin: Dr. B. Smith, ARC Unit of Nitrogen Fixation, University of Sussex, Brighton, U.K. for Bacillus polymyxa; Dr. R. P. F. Gregory, University of Manchester, U.K. for Petroselenum sativum (parsley); Dr. I. Altosaar, University of British Columbia, Vancouver, Canada, for Sambucus sp. (elder); Dr. P. B6gers, University of Konstanz, G.F.R. for Bumilleriopsis filiformis; Dr. R. V. Smith, Freshwater Biological Investigation Unit, Antrim, U.K. for Microcystis flos-aquae and Anabaena cylindrica; Dr, A. Aitken, University of Edinburgh, U.K. for Anacystis nidulans and Anabaena variabilis; Dr. M. C. W. Evans, University College, London for Chromatium and Chlorobium limicola. Apoferredoxins were prepared as described by Hong and Rabinowitz [17]. Concentrations were determined by the Lowry protein estimation.

Preparation of antibodies Antibodies to Beta vulgar& ferredoxin were prepared as described by Tel-Or et al. [18]. Antibodies to Spirulina maxima, Scenedesmus obliquus, Bacillus stearothermophilus and Clostridium pasteurianum ferredoxins were prepared by immunization of New Zealand white rabbits, two for each ferredoxin. 1 mg ferredoxin was suspended in 0.5 ml 10 mM sodium phosphate 0.15 M NaC1, pH 7.4, emulsified with an equal volume of complete Freund's adjuvant (Difco) and injected subcutaneously in multiple sites. The immunization was repeated after 10 days, using the same emulsified

122 material. Sera were collected weekly, starting a week after the second immunization, pooled separately for each rabbit, and tested for interaction with the original ferredoxin by immunoprecipitation. All sera used contained at least 0.5 mg precipitable antibodies per ml. Immunoglobulin fractions (Ig) were separated from each rabbit serum by precipitation with 40 ~o (w/v) ammonium sulphate. The precipitated immunoglobulins were washed twice in 40~o saturated ammonium sumphate in 10 mM phosphate, 0.15 M NaC1, pH 7.4, dissolved finally in 10mM phosphate, 0.15 M NaCI, pH 7.4 and dialyzed against the same buffer (48 h, four buffer changes). An Ig fraction from normal, non-immune rabbit serum was used as a control. The final contents of precipitable antibodies (mg/ml) were: Beta vulgaris 2.5; Spirulina maxima 2.8; C. pasteurianum 2.1; B. stearothermophilus 3.4; Scenedesmus obliquus 1.5; the total protein content of these fractions were 44.6, 46.4, 35.7, 42.9 and 44.6 mg/ml respectively. Therefore, the specific immunoglobulin content of the Ig fractions varied from 3.4~o (Scenedesmus) to 8% (B. stearothermophilus). The same batch of lg fraction of each antiferredoxin from one specific rabbit was used for these comparative studies. The Ig fraction ot anti-Beta vulgaris ferredoxin maintained similar activity when stored at --20 °C from October 1971 until December 1975.

Immunoprecipitation reaction This reaction was carried out essentially as described by Fuchs and Sela [19]. Each reaction mixture contained 0.2 ml of rabbit anti-serum or lg fraction and the ferredoxin to be tested was adjusted to a final volume of I ml with 10 mM phosphate, 0.15 M NaC1, pH 7.4. The concentration of the ferredoxin was determined from the absorbance at 420 nm for the 2Fe-2S ferredoxins (extinction coefficients are approx. 10 mM -1) and 390 nm for the 4Fe-4S ferredoxins (extinction coefficients are approx. 15 mM -1 per 4Fe cluster). Samples were incubated for 1 h at 37 C and left overnight at 4 °C. The precipitate was collected by centrifugation at 11300 ~,~ g for 10 min, washed twice with buffer, then dissolved in 0.1 M NaOH prior to measuring the absorbance at 280 nm. RESULTS

Standardization of the antibody-antigen interaction The immunoprecipitation reaction was found to be very reproducible and exhibited an efficient antigenic resolution between one ferredoxin and another. Prior to the comparative experiments the antibody-antigen saturation curve for each antibody was determined. The optimal concentration for the five ferredoxins for 0.2 ml of the lg fraction were: Beta vulgaris 2 I~M, Spirulina maxima 2 ttM, Scenedesmus obliquus 2 #M, B. stearothermophilus 3/~M and C. pasteurianum 5 t~M. In the comparative studies all five Ig fractions were cross-reacted with 2/~M ferredoxin. This made no significant difference to the comparison, since the antibacterial ferredoxin lg scalcely interacted with nonhomologous ferredoxin. The standard error in the procedure was ± 0.02 absorbance units at 280 nm. All five ferredoxins used fol the immunization were also tested for possible nonspecific interaction with normal nonimmunized rabbit Ig but no interaction was ever detected.

123 TABLE I I M M U N O P R E C I P I T A T I O N CROSS-REACTION OF F E R R E D O X I N S WITH ANTI-FERRED O X I N Ig The immunoprecipitation assay was carried out as described in the Methods section. 0.2 ml of each o f the immunoglobulin fractions was interacted with each of the ferredoxins (2/~M) in a final reaction mixture volume of 1 ml. The values of interaction are expressed as a percentage of the interaction between each immunoglobulin fraction and its homologous ferredoxin. The standard error in this procedure was -+- 0.02 absorbance unit at 280 nm. The 100% values in absorbance at 280 nm were: C. pasteurianum - 0.38, B. stearothermophilus - 0.88, Scenedesmus obliquus - 0.41, Spirulina maxima 0.72, Beta vulgaris - 0.68. Species

Source of ferredoxin to which antibody was produced

Beta vulgaris

Bacterial 8Fe-8S Clostridium pasteurianum Clostridium acidi-urici Chromatium spp. Chlorobium limicola

Scenedesmus

Spirulina

C. pasteurianum

B.

stearothermophilus

8 5 16 9

12 19 19 11

4 5 13 9

1O0 10 4 4

2 5 9 8

Bacterial 4Fe-4S Bacillus stearothermophilus Desulphovibrio vulgaris Chromatium HiPIP

8 7 5

13 13 19

6 9 12

l1 4 6

100 5 7

Blue-green algae Spirulina maxima Spirulina platensis Nostoc strain MAC Type I Nostoc strain MAC Type II Anabaena flos-aquae Anabaena cylindrica Anabaena variabilis Anacystis nidulans Mastigocladus laminosus Microcystis flos-aquae Aphanocapsa 6714 Chlorogloea fritschff

24 24 28 27 32 24 25 19 17 34 26 28

41 34 25 15 32 34 31 21 36 26 23 29

100 96 16 25 46 29 48 36 53 37 27 64

4 6 6 7 8 5 9 4 4 8 6 5

8 5 8 5 5 5 6 3 3 6 0 0

Red algae Cyanidium caldarum Porphyra umbilicalis Porphyridium cruentum

28 15 16

16 15 24

37 43 14

7 8 3

7 4 3

Yellow-green alga Bumilleriopsis fi~formis

12

25

15

6

3

Green algae Scenedesmus obliquus Euglena graeilis

13 21

1O0 30

13 22

6 3

4

16 100 79 37 27 54

20 42 24 29 13 0

16 23 19 20 22 12

6 3 3 6 6 0

6 6 4 2 5 3

Higher Plants Equisetum telmateia (horsetail) Beta vulgaris (Swiss chard) Spinacia oleracea (spinach) Medicago sativa (alfalfa) Petroselenum sativum (parsley) Sambucus spp. (elder)

1

124

General patterns of comparative immunological cross-reactions Table I presents the relative values of antibody-antigen interaction obtained by the immunoprecipitation experiments. It is seen that the bacterial ferredoxins are foreign to each other in their antigenic properties. None of the bacterial ferredoxins interacted with anti-B, stearothermophilus ferredoxin Ig to more than 9 ~,, of the interaction with the original ferredoxin. This ferredoxin has recently been characterized as a four-iron ferredoxin [13] and differs in its physico-chemical properties from the eight-iron bacterial ferredoxin. Since none of the bacterial ferredoxins listed in Table 1 was found to be related to the B. stearothermophilus fertedoxin, we have cross-reacted the anti-B, stearothermophilus ferredoxin Ig with the four-iron ferredoxin I and l I of B. polymyxa which have similar molecular weights and spectroscopic properties to those of B. stearothermophilus ferredoxin [20, 21]. As shown in Fig. 1

1.0

Itus

0.8

A280 0.6

04

02 ^ 0

~ 0

~ 1

ti polyrnyxa n 2

l ~ i ~" B. l~otym~yxa I 3 z, 5 6 Ferredoxin (}JM)

i

7

5

Fig. 1. Precipitation saturation curve o f antibodies to B. stearothermophihtswith ferredoxins o f two Bacillus species. The i m m u n o p r e c i p i t a t i o n reaction was carried out using a constant a m o u n t (0.2 ml) o f anti-Bacillus f e r r e d o x i n l g with increasing a m o u n t s o f ferredoxins f r o m B. stearothermophihlsand the two f e r r e d o x i n fractions o f B. polymyxa (I, ll).

there was little interaction between either of the B. polymyxa ferredoxins with the anti-B, stearothermophilus ferredoxin lg, indicating poor antigenic relationship between the species. Specific variation between ferredoxins of two related bacterial species is also shown by the poor cross-reaction of anti-C, pasteurianum ferredoxin Ig and the ferredoxin of C. acidi-urici (Table I). This result confirms the observation of Hong and Rabinowitz [8]. The plant and algal ferredoxins showed only a very low interaction with both anti-bacterial ferredoxin lg, and the bacterial ferredoxins, exept that of Chromatium ferredoxin cross-reacted very poorly with the plant and algal ferredoxin Ig. This latter finding is somewhat unexpected since, although Chromatium ferredoxin is larger than the other bacterial ferredoxins, it has bacterial-type 4Fe-4S chromo-

125 phores, and would be expected to have a different folding of the polypeptide chain. When the higher plant ferredoxin antigenic properties are compared, as tested in the cross-reaction of plant ferredoxins with anti-Beta vulgaris ferredoxin Ig, a wide variation is found. Ferredoxin from Equisetum (horsetail, a primitive plant) [22] shows the poorest interaction, while Spinacia (spinach) ferredoxin is similar to Beta vulgaris in its interaction with anti-Beta vulgaris ferredoxin Ig. Ferredoxins from the dicotyledonous plants Sambucus (elder), Medicago (alfalfa) and Petroselenium (parsley) showed progressively lower interaction. We have shown previously [10] that the ferredoxin of Zea mays (maize) a monocotyledonous plant gives a low cross-reaction. Plant ferredoxins show a better cross-reaction than the blue-green algal ferredoxins with the anti-Scenedesmus ferredoxin Ig, and are therefore closer antigenically to the green algae than the blue-green algae. It may be noted that cross-reactivity between antigen-antibody pairs is never a mirror image; for example, Scenedesmus ferredoxin does not interact with anti-Beta vulgaris ferredoxin Ig to the same extent as Beta vulgaris ferredoxin with anti-Scenedesmus ferredoxin Ig. In the group of green, yellow and red algae, it is clear that the Scenedesmus ferredoxin exhibited similar antigenic distances from plant and blue-green algal ferredoxins as suggested by the cross-reaction with the Ig to these ferredoxins. The anti-Scenedesmus ferredoxin Ig shows stronger cross-reactions with the yellow-green alga Bumilleriopsis and the green alga Euglena than with the red algal ferredoxins. The three red algal ferredoxins are also clearly differentiated from each other. It may be noted that this distinction is shown by the interaction with three different antibody preparations; it would be less easy to classify them by their interactions with a single antibody. The data for blue-green algal ferredoxins based on the use of 12 strains show first that Spirulina platensis ferredoxin, which differs from Spirulina maxima by four variations in its amino acid sequence [23, 24] interacts almost identically with the anti-Spirulina maxima ferredoxin Ig. Second, both Spirulina ferredoxins have a greater affinity for anti-Scenedesmus ferredoxin Ig than for anti-Beta vulgaris ferredoxin Ig. Among the blue-green algae Chlorogloea fritschii ferredoxin exhibits the closest antigenic resemblance to the Spirulina ferredoxins followed by Mastigocladus. The blue-green algal ferredoxins do not show a systematic tendency of cross-reaction with any one of the three plant and algal ferredoxin Ig fractions, which complicates any clear definition or evolutionary trends, but they do maintain antigenic family proximity with the Spirulina ferredoxin. The two types of ferredoxin which were isolated from Nostoc sp. MAC [14] differ from each other in cross-reaction with Spirulina ferredoxin Ig and Scenedesmus ferredoxin Ig. The three ferredoxins from the three species of Anabaena all seem to be very similar. They react identically with anti-Scenedesmus ferredoxin Ig, but Anabaena cylindrica ferredoxin reacts less well with anti-Spirulina ferredoxin Ig. Although blue-green algae have been alternatively termed "Cyanobacteria" on the basis of their genetic and structural properties, these immunological studies on their ferredoxins clearly link them to the algae.

Amino acid sequence and immunological cross-reaction Margoliash and co-workers [25, 26], in comprehensive studies on the antigenic

126 properties of cytochrome c, have demonstrated that cytochromes with identical primary structure isolated from different species are indistinguishable in their crossreactions with antibodies to three different cytochromes, but that even a single substitution in their amino acid sequence can markedly affect the degree of antigenic cross-reactivity. Since several of the ferredoxins used in our studies have already been sequenced, we investigated the possible effect of change in the amino acid sequences on the antigenic properties of such ferredoxins. Comparative studies of the two Clostridial ferredoxins (C. pasteurianum and C. acidi-urici) showed that these had little antigenic affinity. This is in keeping with the fact that there are 14 variations in amino acid sequence between these small proteins [27] and indicate that some of these variations affect the antigenic determinant sites of the ferredoxins. On the other hand, four substitutions in sequence between Spirulina maxima and Spirulina platensis had no significant effect on the antigenic cross-reactions, suggesting that such substitutions did not involve the antigenic determinant site in either ferredoxin. Since the bacterial ferredoxin and the plant-type ferredoxins are quite different in size and in amino acid sequence, we have investigated a possible correlation between changes in sequence and relative immunological cross-reaction among several of the plant type ferredoxins of which the sequences are known, namely ferredoxins of Spirulina species, Scenedesmus, Spinacia and Medicago. Fig. 2 illustrates the relationship between the degree of immuno-precipitation of these ferredoxins and the number of substitutions in sequences of the the ferredoxins from Spirulina maxima [24], Scenedesmus [28] and Spinaeia [29]. The results obtained with the anti-Scenedesmus ferredoxin Ig show an almost linear relationship between the two parameters suggesting an even distribution of the antigenic determinants in the lo0 7

~ ©

m 50

•

•

"d

0 o

Substituliorls in sequence

Fig. 2. The dependence of immunological cross reaction on n u m b e r of substitutions in the sequence of the ferredoxins. The n u m b e r of substitutions were calculated from the published sequences of Spinaeia, Medicago, Spirulina maxima and Scenedesmus obliquus. The relative values of cross reaction were taken from Table 1: ~.3, anti-Spirulina maxbna ferredoxin lg; O, anti-Scenedesmus obliquus ferredoxin Ig; A, anti-Beta vulgaris ferredoxin lg.

127 Scenedesmus ferredoxin molecule. Similar results were obtained with the anti-Beta vulgaris ferredoxin Ig. The anti-Spirulina maxima ferredoxin Ig interaction on the other hand shows no linear relationship, suggesting an uneven distribution of the immunogenic sites on the Spirulina ferredoxin molecule. The apoferredoxin as an antigen A large amount of data has been accumulated on the immunogenicity of the apoproteins of various globular proteins and on the comparative antigenicity of the apoproteins and holoprotein molecules [7, 8, 30], but such studies on ferredoxins have been restricted to a few bacteria only. In this study we have used the Ig fractions produced against five different ferredoxins to follow the interactions with apoferredoxin prepared from the h o m o logous batches o f ferredoxins used for the immunization and immunoprecipitation reactions. Each of the five ferredoxins and its apoferredoxin were tested in a precipitin saturation curve with a constant amount of Ig, and increasing amounts of the antigens. The results obtained are presented in Fig. 3. The interaction of C. pasteurianum apo10 ( a ) C. P a s t e u r i a n u m 08 06

02-

° l

....

. . . .

L

i

i

i

!

i

I

(c) S p i n a c i a

I

(d) Beta

I

I

i

i

vutgaris

OB

O6 A280 0a

02

* (e)

,

Spirulina

i

i

i

i

i

i

(f) S c e n e d e s m u $

maxima

08

J3,__

06 04

_-O

02 I

I

2

I

4

Concentration

0

6

of

ferredoxin

2 or

apoferredoxin,

4

6

#N1

Fig. 3. Comparative interaction between apo- and native ferredoxins with the anti-native ferredoxin immunoglobulins. Immunoprecipitations were carried out using 0.2 ml immunoglobulin (lg) fraction in a reaction mixture of I ml. The extent of immunoprecipitation is expressed as A280 of the solution after redissolving the precipitate in NaOH solution (See Materials and Methods). Points O--O, native ferredoxin; ©---O, apoferredoxin. (a) C. pasteurianum ferredoxin; (b) B. stearothermophilus; (c) Spinacia oleracea ferredoxin; (d) Beta vulgaris ferredoxin; (e) Spirulina maxima ferredoxin; (f) Scenedesmus obliquus ferredoxin.

128 ferredoxin with the anti-Clostridial ferredoxin Ig (Fig. 3a) is considerably lower than with the protein in agreement with the results of Hong and Rabinowitz [8]. The B. stearothermophilus ferredoxin (Fig. 3b) on the other hand, shows half the degree of interaction of that of the holoferredoxin, indicating that some of the antigenic determinants of this ferredoxin are hidden in the apo-conformation. Fig. 3c and 3d respectively show the saturation precipitin curves of Beta vulgaris apoferredoxin and Spinacia apoferredoxin interacted with the anti-Beta vulgaris ferredoxin Ig. It is seen that while the apo-Beta vulgaris apoferredoxin interacted with the Ig to a similar extent to the native protein, the Spinacia apoferredoxin yielded only one third of the precipitin obtained with the Spinacia holoferredoxin. Thus, two ferredoxins which react similarly in the holo-conformation, behave differently in the apo-conformation. Fig. 3e shows that the Spirulina apoferredoxin gives a lower interaction than the native molecule. On the other hand, Scenedesmus ferredoxin (Fig. 3f) shows a higher precipitation value when cross-reacted with the apoferredoxin than with the holoferredoxin against which the lg was prepared. It is possible that in the case of the Scenedesmus ferrodoxin it became partly converted to the apo-configuration after injection into the rabbit, and that this is more effective in stimulating antibody formation. If this is the case, all the observations of a positive interaction between the Ig and apoproteins should be treated with some caution. However, the differences in interaction between apoproteins and holoproteins are significant. They show that extrusion of the iron-sulphur cluster from the holoproteins leads to changes in the folding of the globular structure which in turn affect the antigenic properties of the molecule. Thus, antigenic properties of even these small proteins do not depend simply on its amino acid sequence, but also on their conformation. In order to help determine whether the cross-reactions shown in Table I represent the total content of the antigenic determinants of each of the heterologous ferredoxins, or whether antigenic determinants which are masked in the holoprotein conformations, may become exposed in the conformation of the apoferredoxins, and thus show additional antigenic activity, each of the five different immunoglobulin fractions was cross-reacted

TABLE II THE RELATIVE INTERACTION OF APO- AND NATIVE FERREDOXINS WITH ANTIFERREDOXIN IMMUNOGLOBULIN 0.2 ml of each of the immunogtobulin fractions were used for the immunoprecipitation assay in a 1 ml reaction mixture with the concentrations of ferredoxins and apoferredoxins which were found to yield maximal interaction in Fig. 3. Units are in absorbance at 280 nm. Antibody

Beta vulgaris Scenedesrnus Spirulina maxima C. pasteurianum B. stearotherrnophilus

Antigen

Spinacia

Scenedesrnus Spirulinamaxima

B. stearotherrnophilus

apo

holo

apo

holo

apo

holo

apo

holo

0.24 0.06 0.07 0.05 0.05

0.60 0.06 0.09 0.05 0.07

0.10 0.74 0.08 0.05 0.07

0.10 0.43 0.09 0.07 0.05

0.14 0.26 0.47 0.07 0.04

0.22 0.16 0.78 0.06 0.06

0.05 0.06 0.05 0.06 0.47

0.05 0.07 0.05 0.04 0.96

129 with the apo- and holoferredoxins of the four species (Table II). The amount of Ig fraction used (0.2 ml) and the concentration of each of the ferredoxins were the same as all previous studies, while the concentrations of the apoferredoxins used were those which yielded optimal interaction with the homologous antibodies. DISCUSSION The main aim of the present study was to obtain additional information on the possible use of immunochemistry in the study of bacterial, algal and higher plant ferredoxins. All five ferredoxins tested were found to be good immunogens, despite their low molecular weights. The first two injections of 1 mg ferredoxin at 10-day intervals were sufficient to induce the immune system of the rabbit to produce antibodies within three weeks from the first injection. Although there were variations in the rate and extent of antibody production for each individual l abbit, we feel safe in predicting that ferredoxins are very good immunogens by the procedure used. It was essential for the comparative studies to use the same batch of pooled serum from one rabbit to obtain a constant population of antibodies for such comparative studies, since it is possible that the immune response may yield a different distribution of antibodies to several antigenic determinants of the ferredoxins in each individual rabbit. The ferredoxin-antiferredoxin interaction is dependent on the primary amino acid sequence and tertiary conformation of the antigen. This is shown by the differences in immunological behaviour between the holo- and apoferredoxins. Although the apoferredoxins reacted less well in general with the anti-ferredoxin Ig (apart from Scenedesmus which appears to be an anomaly), we conclude that the folding of the polypeptide chain of the ferredoxin can lead to either increase, decrease or no change whatsoever in the antigenic activity, depending on whether the antigenic determinants of the protein are masked or exposed. These observations may help to explain the variation in the results obtained in previous studies [7, 8, 30] in which the immunochemistry of apo- and holoproteins of bacterial ferredoxins and rubredoxins were compared. Our attempt to correlate the experimental antigen-antibody interaction data with the amino acid sequence of the ferredoxins showed a relationship between primary structure and antigenic properties of the ferredoxin molecule. The determination of more sequences of algal and plant ferredoxins might further clarify this relationship. The antibodies to Clostridium and Bacillus ferredoxins showed very little antigenic cross-reaction with two species of the same genus, and such heterogeneity seems to rule out the possible use of antibodies to ferredoxins in bacterial taxonomy. However, in the immunoprecipitation assay we were unable to detect monovalent antigen-antibody interactions which do not lead to precipitation and, as was already shown by Yasunobu and Lovenberg [30], such an approach was essential to detect relationships between bacterial rubredoxins. Possibly the bacterial ferredoxins may overlap in only one antigenic determinant; this requires further investigation. The poor cross-reaction with the bacterial ferredoxins precludes any recognition of evolutionary trends between the bacterial species used and the higher

130 organisms, with the possible exception of an interaction between Chromatium ferredoxin and antibodies to the plant and algal ferredoxins. When the algal ferredoxins are considered, results are more promising. It was very easy to detect, for example, the close taxonomic relationship between the two species of Spirulina and to distinguish the two types of ferredoxin isolated from Nostoc MAC. Clearly, antibodies can be used as auxiliary taxonomic markers in addition to other biochemical parameters. From the evolutionary point of view, our studies do not suggest a clear main trend of evolution of the plant type ferredoxins. However, they do provide a quantitative measure of the relationship between algal and plant ferredoxins with a definite power of resolution. It is easy to detect relations within each group of ferredoxins and to follow the interrelation between blue-green and green algae, or blue-green and red algae. A more comprehensive collection of antibodies might make possible a more detailed investigation of the relationship between algal and plant ferredoxins. Our comparative results also show that attempts to measure concentrations of ferredoxins from various origins by an antibody to only one of them, should be approached with caution. Such a method has been proposed recently [11] for the quantitative determination of algal and plant ferredoxins. The different content of ferredoxin estimated by these investigators may arise from the different binding capacity of the antibodies for each of the antigens used. We believe that the immunochemical investigations of non-haem iron proteins provide a powerful potential for the understanding of the structural chemistry, taxonomy and evolution of this important group of proteins. ACKNOWLEDGEMENTS We thank Dr. D. W. F. Wheater for his helpful supervision of the work with the rabbits, and Dr. R. N. Mullinger for assistance with the preparation of the apoferredoxins. This work was supported by the Science Research Council. We also thank the European Molecular Biology Organization for a short-term fellowship to E.T.O. which initiated these studies. REFERENCES

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