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Studies on the antigenic properties of ferredoxin from Clostridium pasteurianum Nitz, Rodney Marcus 1970

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STUDIES ON THE ANTIGENIC PROPERTIES OF FERREDOXIN FROM CLOSTRIDIUM PASTEURtANUM by RODNEY M. NITZ -B.Sc. Microbiology University of British Columbia A THESIS SUBMITTED IN PARTCAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology as conforming standard We accept this thesis to the required THE UNIVERSITY OF BRITCSH COLUMBIA 25 March, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia Vancouver 8, Canada ABSTRACT It was established that antibodies could be evoked in rabbits against ferredoxin purified from cultures of Clostridium pasteurianum and against its performic acid oxidized derivative. The extent of cross-reaction was studied between the two antisera and four related antigens: native ferredoxin, iron-sulfide free ferredoxin, performic acid oxidized ferredoxin, arid S-carboxymethylated ferredoxin. All combinations demonstrated cross reactivity by complement fixation, and in the case of oxidized ferredoxin antiserum, three preparations, native ferredoxin, iron sulfide free ferredoxin, and performic acid oxidized ferredoxin precipitated antibody. The data obtained with these cross-reactivity studies Indicated that the cysteine-containing regions of the ferredoxin molecule were not crit ically involved as antigenic determinants. The C-terminal region of the protein was considered for further study. This octapeptide was synthesized and tested for its ability to combine with antibody directed against both native ferredoxin and its performic acid oxidized derivative. The peptide exhibited specif binding to both antisera as demonstrated by inhibition of complement fixation and precipitation, and by equilibrium dialysis experiments. It Is suggested that C_. pasteurianum ferredoxin is antigenic in rabbits, that cysteine residues are not involved in at least two of the antigenic regions of the protein, and that the C-terminal octa-peptide is one of the antigenic determinants of this molecule. TABLE OF CONTENTS Page INTRODUCTION AND LITERATURE REVIEW . 1 I. Determination of Antigenic Sequences 2 II. Importance of Configuration 11 III. Nature of the Antigenic Site I'* MATERIALS AND METHODS 18 I . Cul tures 18 II. Purification of Ferredoxin • • • • 20 III. Analytical Procedures 22 IV. Chemical Modification of Ferredoxin 23 V. Immunization Procedure 24 VI. Complement Fixation . 25 VII. Quantitative Precipitation Reaction 27 VIII. Peptide Synthesis 28 IX. Hapten Inhibition Studies 29 X. Equilibrium Dialysis 31 RESULTS AND DISCUSSION . 35 1. Preparation of Ferredoxin 35 II. Antisera 0^ III. Modification of the Cysteine Residues of Ferredoxin • • • • kl IV. Immunological Reactivity of Ferredoxin and its Derivatives hi I V Page V. Solid Phase Synthesis of the COOH-Terminal Peptide 51 VI. Immunological Testing of the Synthetic Peptide . . . Sk CONCLUDING REMARKS . . . . . . 68 LITERATURE CITED 70 LIST OF TABLES v Page Table 1. Yield of cell paste and ferredoxin with the 390/280 my ratios of the four batches purified . . 36 Table 2. Amino acid compositions,of the various antigenic preparations used in this study. 39 Table 3- Amino acid analysis of the C-termtnal octapepttde of ferredoxin 56 Table k. Equilibrium dialysis data . 66 vi LIST OF FIGURES Page Fig. 1. Studies on the Antigenic Sites of Sperm Whale Myoglobin 5 Fig. 2. Tertiary Structure of Sperm Whale Myoglobin. . . . 6 Fig. 3. Amino Acid Sequence of Hen-Egg White Lysozyme. . . 10 Fig. h. Spectrum of C_. pasteurianum ferredoxin 38 Fig. 5- Quantitative precipitation reactions of 0 Fd Antiserum against Various Antigenic Preparations' kl Fig. 6. Amino Acid Sequence of C_. pasteurianum ferredoxin hk Fig. 7- Complement Fixation Reactions of Antiserum Evoked Against Native Ferredoxin 48 Fig. 8. Complement Fixation Reactions of Antiserum Evoked Against 0 Fd kS Fig. 9. Flow Chart of Solid Phase Peptide Synthesis. . . . 53 Fig. 10. Elution Profile of the C-terminal Octapeptide. . . 55 Fig. 11. Inhibition of Complement Fixation by the C-terminal Octapeptide 58 Fig. 12. Inhibition of Precipitate With 0 Fd antiserum and its Homologous Antigen in the Presence of the COOH terminal Octapeptide 60 Fig. 13. The Elution Profile of the NH^Acetylated Octapeptide 61 v i i ACKNOWLEDGEMENTS I would like to thank Dr. Julia Levy for her many suggestions, helpful criticism, and invaluable encouragement during the research and writing of-this thesis. I also wish to thank Dr. H. Whitely for kindly supplying cultures of Clostridium pasteurianum and for her suggestions concerning the culturing of the organism. And I wish to extend my gratitude to Mrs. Barbara Mitchell and John Christensen for their cooperative contributions and to Mrs. Barbara Mitchell for drawing many of the figures used in this thesis. Finally, I extend thanks to Dr. Doug Kilburn and Dr. Tony Warren for their suggestions concerning the final draft. INTRODUCTION AND LITERATURE REVIEW Until recently the antigenic determinants of naturally occurring proteins have been determined for only a few proteins and within rather broad limits; certainly the smallest sequence of amino acids in a protein capable of binding with antibody directed against the whole antigen has been determined for only one protein, that of tobacco mosaic virus. Molecular studies on peptide antigens have been carried out using mainly synthetic homopolymers or heteropoly-mers of known composition,.though not necessarily known sequence. Many reviews of this work have been published in the last five years (Kabat, 1 9 6 6 ; 1 9 6 8 ; Sela, 1 9 6 6 ; G i l l , Kunz, Freedman and Doty, 1 9 6 3 ; and Proceedings of the Royal Society, Series B, 1 9 6 6 : Gabe, Richards and Sela). The work' is extensive and most conclusions refer to general properties of the antigenic determinants such as charge, size, conformation, and chemical properties. These will be dealt with only insofar as they relate to the work discussed in this thesis. In order to obtain useful information concerning the immune response to naturally occurring proteins, the sequence of peptides found to bind antibody directed against the whole molecule should be placed within the context of the entire sequence and structure of the protein. Lately, the amino acid sequences arid conformations of many proteins have been determined, making such a study feasible. 2 The ferredoxin molecule of C_. pasteurjanum is especially suited to such a study.' It is easily purified in suitable quantities; it is a small protein (MW 5500), with few aromatic residues 0 phenylalanine and 1 tyrosine); it has a single basic residue (lysine), making it extremely electronegative; and it contains eight cysteine residues, relatively evenly spaced in groups of.four along its sequence, with no disulfide bridges,' lessening the chance of conformational variables. Also, ferredoxins from several sources have been purified and their sequences established. They all demonstrate a high degree of sequence homology (Tsunoda and Yasunobu, 1968), and provide a naturally occurring series of antigens which can be studied for their cross-reacting properties, and ultimately for relationships of antigenic sites from different species. This thesis presents initial studies on determining the antigenic determinants of ferredoxin from C1ostridiurn pasteurianum. I. Determination of Antigenic Sequences The principle method employed for the determination of the antigenic determinants of naturally occurring proteins involves the isolation of peptides from the parent molecule and the subsequent testing of their ability to bind with antibody produced against the whole molecule. Although a number of globular protein antigens, such as bovine serum albumin (Porter, 1957), human serum albumin 3 (LaPresle et_ aj_, 1957, I960), diptheria toxin (Raynaud e_t a_l_, 1955) and thyroglobul in (Metzgar et_ a_l_, 1962), yielded fragments by enzyme hydrolysis which were capable of combining with antibodies evoked by intact antigens, these fragments were of high molecular weight and appeared to have more than a single antigenic determinant (polyvalent) since they gave precipitates with homologous antisera. Press and Porter (1962) isolated a chymotryptic peptide from human serum albumin which caused 50% inhibition of precipitation with the homologous system when tested at a molar concentration 1.5 times that of the intact antigen. This peptide did not precipitate anti-HSA serum by itself. The molecular weight of the fragment was estimated to be 7100 and no attempt was made to elucidate its chemical structure, aside' from its amino acid composition, or to determine if a smaller portion of the peptide was capable of binding ant i body. An octapeptide of composition gly(gly^ala^) tyr which was isolated from silk fibroin by Cebra (1961) inhibited precipitation in the homologous system by kO to 90%, depending on the serum used. Glycine, alanine, and tyrosine compose h5.S%, 26.k% and 5.6% respect-ively of the fibroin molecule (Florkin and Stotz, 1963). Removal of the C-terminal tyrosine with carboxy-peptidase caused a 50% decrease in the peptide's binding to anti-fibroin serum. No attempt was made to locate the peptide in the fibroin molecule. Brown (1962) isolated active peptides from oxidized bovtne r i bonuclease arid found one peptide, encompassing residues 39 to 6 2 , to be 50% as active as the intact antigen in inhibiting specific precipitation, while not precipitating antibody itself. No attempt was made to find the smallest peptide in this sequence capable of binding antibody directed against the whole molecule. Four chymotryptic peptides, ranging in length from 7 to 19 residues, were shown by Crumpton and Wilkinson (1965) to inhibit precipitation between metmyglobin (containing haem) and its homo-logous antiserum, while they did not precipitate antibody themselves (Figure 1 ) . Atassi and Saplin (1968) isolated 5 tryptic peptides from sperm whale myoglobin which inhibited precipitation of the whole molecule by its homologous antiserum, while they had no precipitat-ing activity themselves. These peptides are also shown in Figure 1. It is interesting to note that four of these peptides occupy corner positions in the three-dimensional model of the protein, while the fifth is the C-terminal hexapeptfde (Figure 2 ) . They also found that cyanogen bromide cleaved the protein at the methionine residues at positions 55 and 131 , producing three fragments: the first two fragments ( 1 - 5 5 and 5 6 - 1 3 1 ) formed immune precipitates', and each contained two of the five tryptic peptides found to be antigenic; the third fragment showed inhibitory activity only, and was found to contain the last of the active peptides. Together the three fragments produced by cyanogen bromide (CNBr) cleavage accounted for 100% of the precipitating capacity of the antisera. Atassi 5 V a l 1 T r p 7 ( A l a 1 5 - V a l 1 7 A r g 3 1 — P h e 3 3 ) Lys3k S e r 3 5 Phe1*3 M e t 5 5 ( L y s 5 6 L e u 6 9 ) T h r 7 ° — L y s 7 9 L y s 9 6 - H i s 9 7 T ^ r 1 0 3 , T . 19 „ V 4.131 „ 132 His Met — Asn C h 140 *1^6\ i f l 1U7 „, 1^ 8 * 1 5 l _ p , 153^ (_Arg Tyr J Lys - Glu Tyr Gly J Figure 1. Studies on antigenic s i t e s of Sperm Whale Myoglobin. ( ) - enclose haptenic chymotryptic peptides (Crumpton & Wilkinsoa, 1965) I | - underline haptenic t r y p t i c peptides (Atassi & S a p l i n , 1968). y ~ cleavage points, of Cyanpgen Bromide ( A t a s s i , 196?). % - n i t r a t e d Tyrosines ( A t a s s i , 1968). Figure 2 . T e r t i a r y structure of Sperm Whale Myoglobin (Atassi & S a p l i n , 1 9 6 8 ) . The approximate: position'of reactive t r y p t i c ' p e p t i d e s are shown by marking on the main l i n e . 7 and Saplin also found that cleavage of the N-terminal heptapeptide at tryptophan 7 left the molecule immunologically intact, in agree-ment with Crumpton and Wilkinson (1965) who found this peptide unreact ive. The results of the work involving separation of antigenic peptides from sperm whale myoglobin are summarized in Figure 1. Of the two areas of correspondence between the results of Crumpton and Wilkin-son (1965) and those of Atassi and Saplin (1968), one of them, residues 17_31, is found in a corner piece between two helical regions, and thus could be more available to the antibody synthesizing mechinery; the second area of correspondence involves the C-terroinal residues, which, in the three-dimensional model, also appear readily available (Figure 2). In each report the complete chymotryptic and tryptic digests gave no more than 25% inhibition, while the 3 fragments obtained by Atassi and Saplin with CNBr cleavage accounted for all the reactivity of the intact antigen. It is likely that some determinants were split by the proteolytic enzymes, "or that isolated peptides underwent extensive configurationa1 changes which prohibited proper alignment for binding to antibody. Chemical modification of specific residues ih myoglobin supports the data found using isolated peptides. Atassi C1967) found that oxidation of the two methionine residues with periodic acid had no effect on the immune reactivity of the whole molecule, indicating 8 that these two residues are not involved in antibody binding. Atassi (1968) also investigated the role of tyrosine in the antigenic 103 reactivity. Nitration at Tyr of the fragment 56-131 obtained by CNBr cleavage, did not reduce reactivity compared with the un-nitrated control. Nitration of tyrosines 146 and 151 in the fragment 132-153 caused complete loss of reactivity. As Figure 1 shows, 103 Tyr is not found in a reactive peptide, while tyrosines 146 and 151 are. Antigenic peptides have been isolated from hen egg-white lysozyme which are capable of inhibiting homologous prectpitat ton with the whole molecule. This protein, too, is an excellent subject for such a study, since its amino acid sequence and tertiary structure have been estab1ished (Canfie1d, 1956 and Blake, 1965)- Shinka et a 1 (1967) isolated four peptic peptides capable of inhibiting precipi-tation by 25% while not precipitating antibody alone. All four peptides were found to cover the sequence Glutamine^^ to Alanine'^ with different single peptide bond breaks at residues 83 and 84 in a loop formed by a disulfide bridge (Figure 3). One of these peptides (with a break at Leucine ) was hydrolyzed further with trypsin, and one peptide, encompassing residues Tryptophan^ and Lysine"^, was recovered which delayed the development of turbidity in the homo-logous precipitation reaction. No attempt was made to degrade the peptide further or to determine the effect of disrupting the disulfide bridges. Fujio et al (1968) isolated a peptic peptide encompassing 9 the N-terminal residues, Lysine to Asparagine , joined to the C-122 129 terminal residues, Alanine to Leucine , by a disulfide bridge at position 127, which he found to possess specific inhibiting power (Figure 3). Atassi and Habeeb (19&9) studied the effect of modifying Tyrosines 20 and 23 on the antigenicity of hen egg-white lysozyme. They found that nitration of these two residues decreased the ability of the molecule to precipitate homologous antiserum by 10 to 33% depending on the serum used. Th|s inhibition is in fair agreement with that found by Atassi (196/) when .sperm, whale myoglobin.was.nitrated. Reduction of the nitrotyrosines to aminotyrosine brought about an almost complete recovery of the antigenic activity. On nitration of tyrosine ortho to the phenolic hydrdxyl group, the electron with-drawing nitro group increases the acidity of the phenolic hydroxy!, while reduction causes a reversal of this effect (pKa Tyrosine = 10.1, pKa N02-Tyr = 7.2, pKa Nr^-Tyr = 10.0). The two tyrosines occur in the N-terminal portion of the peptide found by Fujio et_ aj_ (1969) to be antigenic. In a study on reduced and carboxymethy1ated lysozyme, Gerwing and Thompson (1969) isolated a single tryptic peptide encompassing Ik 96 the sequence Asparagine to Lysine which gave a maximum inhibition of precipitation of 60%. This sequence falls within the peptide found by Shinka et_ a_l_ (1967) to be antigenic. Removal of the three N-terminal amino acids of the peptide completely destroyed its inhibitory Figure 3. Amino acid sequence of Hen Egg-White Lysozyme "(Shinka et a l , 1967). 11 activity, while a synthetic decapeptide covering the sequence Asp"aragine 8k to Leucine was found to be inhibitory. Correlation between this work and that done by other workers on native lysozyme is of doubtful value considering the drastic ahanges in configuration caused by breaking the disulfide bridges (there is no cross-react ion between native lysozyme antiserum and CM-lysozyme). No attempt has been made as yet, to determine if a smaller sequence than the decapeptide is capable of binding with anti-CM-lysozyme antisera. II. Importance of Configuration The importance of configuration in antibody binding has been studied to a limited extent with respect to naturally occurring protein antigens. Spragg et_ aj_ C1968) studied the effect of single amino acid substitutions in the nonapeptide bradykinin with antibody evoked against the peptide linked to a polylysine carrier. Alanine was substituted for different amino acids in the peptide, and the effect of the substitutions on antibody binding was tested. The results indicated that alterations in charge or hydrophobicity-(Alanine for Arginine, Serine or Phenylalanine) had l i t t le effect on binding, while substitutions causing greater freedom or restriction on conformation (Alanine for Glycine or Proline) caused a great reduction in binding. Work on globular proteins shows, generally, that methods which alter conformation, such as disruption of disulfide bridges, destroy reactivity with antiserum evoked against the native protein. Brown (1959) showed that performic acid oxidized ribonuclease did not cross react with native ribonuclease antiserum, and similar results were observed by Thompson and Gerwlng Cl969) employing reduced and carboxymethy1ated hen egg-white lysozyme and antiserum directed against the native protein. Merrigan e_t aj_ ( 1 9 6 6 ) extended the work on the role of con-formation in the antigenicity of ribonuclease, and found that de-naturation by heat or urea completely destroyed the ability of RNase S (obtained by cleavage between residues 20 and 21 with the NH -^termina1 fragment attached to rest of the molecule by a disulfide bridge) and RNase S protein (RNase minus peptide 1-21) to form precipitates with antibodies directed against native RNase in agar gel. Under normal conditions these two derivatives produce reactions of partial identity, indicating that they retain some antigenic sites found in the native molecule, while they are incapable of binding all antiribonuc1 ease antibody. A similar study was carried out by Bonavida et_ aj_ ( 1 9 6 9 ) on hen egg-white lysozyme. They cleaved the two methionine residues in the molecule at positions 12 and 105, but the three peptides formed are held together by disulfide bridges, so the molecule remains intact. The cleaved molecule was found to Inhibit the native system to a maximum of 70% and showed partial identity with native hen egg-white lysozyme antiserum in agar gel, Indicating the loss of some antigenic determinants. Changes In conformation were Indicated by a red shift in the ultra-violet spectrum of the altered molecule, consistent with tryptophan being found in a more hydrophobic env i ronment. Arnon and Sela (1969) studied the dependence of antibody specificity on conformation using the "loop peptide" of hen egg-white lysozyme, which encompasses residues 64 to 83 of the protein. They isolated the peptide from a peptic digest of lysozyme and coupled it to a synthetic copolymer of alanine and lysine. Antibodies directed against this conjugate were raised in rabbits. Purified "loop" peptide was found to inhibit the conjugate precipitin system 75% and reduced and carboxymethylated "loop" was almost as effective-native lysozyme was also found to be an inhibitor of this system to the extent of 35%. The purified "loop" peptide was capable of inhibiting the lysozyme-anti-lysozyme system 5%, while the reduced and carboxymethylated "loop" was inactive. The anti-"loop" antibodies obtained with lysozyme on cross immumoabsorption could discriminate between the peptide s t i l l containing a disulfide bridge and the same peptide after the bridge was opened, to a much greater extent than the anti-"loop" antibodies purified from sera directed against the synthetic polypeptide conjugate. It appeared to the authers that the anti-"loop" antibodies are directed against a conformation dependent determinant, the loop being a more rigid conformation within native lysozyme than when attached to the conjugate. Finally, Crumpton and Small (1967) compared the conformation of two active peptides obtained from sperm whale myoglobin and their e f f e c t s on a n t i b o d y b i n d i n g . In s o l u t i o n , one o f the p e p t i d e s , B.l , c o n t a i n s 14 amino a c i d r e s i d u e s o f which |;2 a r e h e l i c a l i n the n a t i v e m o l e c u l e . The o t h e r , C l a , c o n t a i n s 13 amino a c i d s , o f which 8 i n the m i d d l e a r e n o n - h e l i c a l . O p t i c a l r o t a r y d i s p e r s i o n s t u d i e s i n d i c a t e d t h a t both p e p t i d e s were e s s e n t i a l l y n o n - h e l i c a l i n s o l u t i o n , y e t b o t h were found t o b i n d w i t h a n t i b o d y evoked a g a i n s t the n a t i v e m o l e c u l e . The a u t h o r s suggest t h a t i n s o l u t i o n t h e p e p t i d e s p o s s e s s a v a r i e t y o f c o n f o r m a t i o n s i n c o n t i n u o u s i n t e r c h a n g e and t h a t a n t i -b o d i e s r e a c t w i t h t h o s e m o l e c u l e s whose c o n f o r m a t i o n s a r e t h e same as t h a t o f t h e c o r r e s p o n d i n g r e g i o n i n the n a t i v e p r o t e i n ; the o p t i c a l r o t a r y d i s p e r s i o n s t u d i e s i n d i c a t e d t h a t a s m a l l p o r t i o n o f the m o l e c u l e s o f p e p t i d e Bl may indeed p o s s e s s the h e l i c a l con-f o r m a t i o n i n aqueous s o l u t i o n a l t h o u g h i t c o u l d be no more than 5%. I f t h i s i n t e r p r e t a t i o n i s c o r r e c t , i t i s p o s s i b l e t h a t the h e l i c a l c o n f o r m a t i o n o f p e p t i d e Bl i s s t a b i l i z e d by i n t e r a c t i o n w i t h a n t i b o d y . I I I . N a t u r e of the A n t i g e n D e t e r m i n a n t The most d e t a i l e d s t u d y t o dat e on n a t u r a l l y o c c u r r i n g p r o t e i n s has been c a r r i e d out by B e n j a m i n i and c o - w o r k e r s on t o b a c c o m osaic v i r u s p r o t e i n . They found a s i n g l e t r y p t i c p e p t i d e , c o n t a i n g 20 amino a c i d s , t o have s p e c i f i c h a p t e n i c a c t i v i t y ( B e n j a m i n i e t a l " , 1964, 1965). Removal o f 5 amino a c i d s from the N r ^ - t e r m i n u s o f the p e p t i d e and 2 from the COOH-terminus, produced a t e t r a d e c a p e p t i d e w h i c h was capable of binding anti-TMVP antibody, although binding was somewhat reduced compared to the eicosapeptide (Young, et_ a_l_, 1966) . Stewart et_ aj_ (1966) synthesized the COOH-terminal decapeptide portion of the eicosapeptide and demonstrated that it possessed haptenic activity. Following this, a series of peptides of increas-ing length from the COOH-terminal end of the decapeptide was synthesized and tested for binding activity. It was found that anti-TMVP antibody bound the pentapeptide to the nonapeptide, while the d i - , tri—, and etrrapeptides lacked significant activity. The sequence of amino acids in the pentapeptide was Leu-Asp-Ala-Thr-Arg. The minimum size of 5 amino acids is in close agreement with the results of other studies. Arnon et_ aj_ (1965),.studying rabbit serum albumin conjugated with polylysine peptides of an average chain length of 5-5 residues, found that polylysine peptides showed inhibitory power in the homologous system which increased sharply up to penta-lysine and then levelled off rapidly up to the nonapeptide. Antibodies directed against the poly-D-g1utamic acid capsule of Bacillus anthracis were shown to bind maximally with hexa-poly-D-glutamic acid in the case of two antisera, and with the pentapeptide in a third case (Goodman e_r^  aj_, 1968) . Kabat (i960), studying antibodies against dextran, found that a hexasaccharide was the minimal antigen size capable of binding. The dimensions of hexa-glutamtc acid are 36 x 10 x 6 R and those of isoma 1tohexaose, 3^  x 12 x 7 8. The similarity of the size of the two determinants is especially significant since they were obtained from very different biological substances. Having isolated the smallest sequence of amino acids of the TMV protein eicosapeptide capable of binding antibody elicited against the whole protein, Benjamini et_ a_l_ Cl968) investigated the properties of the peptide that were responsible for specific binding. They found that the coupling of octanoic acid to the N^-terminus of the COOH-terminal t r i - and tetrapeptides caused them to exhibit specific hapten' activity, while the octanoyl dipeptide was inactive, indicating the importance of the hydrophobic character of the t^^-terminal amino acids. To study the relative importance of the 5 amino acids in specific binding, Young et_ a_j_ (1967) synthesized a number of analogues of the peptide. The importance of.the NH2~terminal hydrophobicity was,established, since D- or L-alanine, isoleucine or tyrosine could be substituted for leucine with retention of activity. It appeared that the shape, rather than the negative charge of the aspartic acid, at position k from the COOH-terminus, was important since asparagine but not glutamic acid could be substituted, with conservation of haptenic activity. A peptide of sequence Thr-Leu-Asp-Ala-Thr was inactive, while a peptide of sequence Asp-Ala-Thr-Arg-Arg was active. Threonine and arginine occur at the HH^~ and COOH-terminal ends of the active pentapeptide in the whole TMV protein; the arginine-arginine bond is split by trypsin during isolation of the eicosapeptide. These results suggest the importance of hydrophobic interactions in the binding between antibody and antigen and also serve to emphasive that results obtained with isolated peptides obtained,by enzymic hydrolysis must be interpreted with care, since antigenic sites may be fragmented by the action of the enzyme. With a defined antigenic determinant, the workers were able to compare the specificity of antibodies obtained from different animals While none of the antisera bound the COOH-terminal d i - , t r i - or' tetrapeptides, sera produced by some rabbits bound the pentapeptide while others required the hexa- or heptapeptide for demonstrable binding. The amount of pentapeptide bound by antibodies produced by a given rabbit did not necessarily correlate with the amount of decapeptide bound by antibodies produced by the same rabbit. However it was found that the octanoyl-pentapeptide was bound by all sera tested, including those that did not bind the unsubstituted penta-peptide. The effect was specific for antl-TMVP serum since anti-lysozyme serum showed no binding. The authors have suggested that the octanoyl group enhances a possible hydrophobic interaction between the antigenic area and the antibodies (Benjamini et a 1, 1969) which stabilized the complex, while specificity is determined by the pentapeptide region. MATERIALS AND METHODS Cultures (a) Organism The culture of Clostridium pasteurianum used in this study was kindly supplied by Dr. H. Whitely, Department of Microbiology, University of Washington, Seattle. It was the same strain used by Tanaka et_ aj_ (1966) to obtain ferredoxin for the sequencing of the protein. (b) Stock Cultures Stock cultures were maintained in the following medium (Whitely, H., personal communication, 1969) : peptone, 1%; beef extract, 1%; yeast extract, 0.5%; sodium acetate, 0.5%; Cysteine-HC1 , 0.5%; soluble starch, 0.1%; and agar, 0.2%. The medium was dispensed in 20 ml quantities to 19 -X 150 mm tubes, closed with metal caps and autoclaved for 15 min at 15 lb pressure. Just prior to inoculation the medium was immersed in boiling water for 15 min and rapidly cooled. A 10% inoculum was used for all culturing procedures and incubation was carried out at 30 C for 18 hr. The cultures were stored at k C and subcultured every 3 months. (c) Batch Cultures Organisms from which ferredoxin was purified were grown in 30 litre quantities in 8 gallon stainless steel milk cans. Cultures were grown in the synthetic medium of Lovenberg ( 1 9 6 3 ) , with (NHj^SO^ as nitrogen source. The 30 litres of medium were prepared by dissolving the constituents in 27 litres of tap water in the. milk can. Sterilization was accomplished by bubbling steam "through the medium for 1 hr, by way of'a 1.5 cm diameter stainless steel tube attached to the lid of the can and which extended to within 10 cm of the bottom of the container. At the end of the sterilization time, the medium, which had increased to 30 litres in volume, was cooled rapidly to 30 C in running tap water. The inoculum for batch cultures was prepared by serially inoculating from a stock culture to 250 ml of the maintenance medium and then to 3 litres of the synthetic medium which was used as inoculum for the batch culture. The batch culture was incubated for approximately 18 hr at 30 C. (d) Harvesting The cells from the 33 l itre batch cultures were harvested by continuous flow centrifugation using an 8-tube Szent-Gyorg1-Blum continuous flow apparatus (Ivan Sorvall Co.) in a Serval RC2 refrigerated centrifuge. Cells were sedimated at 30,000 x g at 0 C, with a flow rate of 150-200 ml/min. To decrease foaming during harvesting, 5.0 litre portions of the medium were degassed under vacuum for 15 min and 0.2 ml of tributyl citrate was added as a defoaming agent, prior to centrifugation. The harvested cells were placed in a beaker and held at -10 C overn ight. i i . Purification of Ferredoxin Ferredoxin was purified from the frozen and thawed cell paste according to the method of Mortensen (J964) as modified for a cell paste by Lovenberg and Sobel (1965). The thawed cells were sus-pended in 600 ml of distilled water at k C and stirred with a magnetic stirrer for 1 hr. Then an equal volume of acetone at k C was added and stirring was continued for 5 min at h C. The acetone slurry was cl ar i f i ed by centrifugation at 20,000 x g_ for 30 min ' at h C. The sedimented material was discarded and the ferredoxin was further purified from the supernatant by column chromatography. DEAE cellulose (Biorad Co.) was repeatedly clarified to remove fines and poured into a 25 mm diameter column to a height of 50 mm. The column was equilibrated with 1.0 M phosphate buffer at pH 6.5 and then washed with 500 ml of distil led water. The 50% acetone cell extract was run through the column with a flow rate of 10 ml/min. The yellow column effluent was discarded. The column was then washed with 250 ml of distil led water to remove any acetoney and then with v0.15 M Tris-HCl buffer pH 8.0 until the effluent had an absorbance at 260 my., 1.0 cm light path of a less than 0.10. The ferredoxin was eluted with 0.65 M NAC1 buffered with 0.15 M Tris-HCl pH 8.0 in as small a volume as possible and was desalted by dialysis against 0.015 M Tris-HCl buffer pH 7.3 for 6 hr at h C. Final purification was achieved by ammonium sulphate precipita-tion. Some.contaminating-material was removed from the desalted ferredoxin solution by addition of solid (NH^J^SO^ to 60% saturation. Precipitation was allowed to occur at k C for 6 hr and the preci-pitate was removed by centrifugation at 20,000 x g_ for 20 min. The ferredoxin was precipitated by increasing the (NH^^SO^ concentration to 30% saturation by addition of solid salt. Precipitation was allowed to occur overnight at 4 C, and then the precipitate was re-covered by centrifugation at 20,000 x g_ for 20 min. The reddish supernate was discarded. The precipitate was dissolved 'In a minimum of 0.015 M Tris-HCl buffer pH 7-3, and dialysed against 1 l itre of the same buffer for 10 hr with stirring at k C. The dialysate was changed frequently during this time. The 290/280 my absorbance ratio of the desalted preparation was determined using a Beckman DBG spectrophotometer; and the ferredoxin was. repeatedly preci-pitated with ammonium sulphate at 30% saturation until the ratio ceased to increase. Yields were calculated using the extinction coefficient at 390 my of EJ* =33.2 with 0.015 M Tris-HCl buffer as diluent, as described by Mortenson (1964). II. Analytical Procedures (a) Spectral Analysis The absorption spectra from 200 mu to ^50 mu of the purified preparations of ferredoxin were determined on a Unicam Recording Spectrophotometer Model SP 800 B with a Model SP 20 recorder, using a 1 cm light path, and 0.15 M Tris-HGil buffer as diluent. Cb) Amino Acid Analysis All preparations of ferredoxin and its modified derivatives were checked for their amino acid"compos it ion. Samples were pipetted into glass ampoules and an equal volume of 12 N HC1 was added. The ampoules were sealed under vacuum and hydro-lysed for 18 hr at 110 C. The hydrolysates were washed three times and taken to dryness on a rotary evaporator, dissolved in 1.0 ml of starting buffer and analysed on a Spinco Model 220 amino acid analyzer according to the method of Spackman et_ a]_ (1958) . (c) High Voltage Electrophoresis High voltage electrophoreses were carried out for hS min-utes on a Gilson High Voltage E1ectrophorator in pyridine-acetate buffer (10%; 0.k%) pH 6.5 on Whatman #3 f i l ter paper. Peptide material was located by dipping the paper in a ninhydrin solution prepared as follows: (f) 1% ninhydrin (Pierce Chemicals) in acetone (ii) 5 g cadmium acetate, and 250 ml acetic acid dissolved in 500 ml distil led water (ffi) 85 ml of ( 1 ) and 15 ml of (ii) were mixed just prior to use and the electrophoretogram was developed at room temperature in a fume hood. Chemical Modifications of Ferredoxin (a) Iron Sulfide Free Ferredoxin (TCA-Fd) Iron sulfide free ferredoxin was prepared according to the method of Tanaka et_ a]_ (1964). Thirty mg of ferredoxfn were dissolved in 4.0 ml of distil led water and 2.0 ml of a 15% solution of trichloroacetic acid and 0.1 ml of 2-mercapto-ethanol were added. The mixture was allowed to stand 1 hr at 4 C. The mixture was sedimented in a clinical centrifuge and the precipitate (TCA-Fd) was washed consecutively with 5.0 ml of distil led water, 5.0 ml of 95% ethanol and 5.0 ml of ether. The final precipitate was dried under vacuum. (b) Performic Acid Oxidized Ferredoxin (OFd) The cysteine residues of iron sulfide free f e r r e d o J i c i n were converted to cysteic acid by oxidation with performic acid. Fifteen mg of TCA-Fd were dissolved in 1.0 ml of concentrated f o r m f c a c i d . To t h i s s o l u t i o n was a d d e d 2.0 ml o f p e r f o r m i c a c i d ( p r e p a r e d by m i x i n g 9.0 ml o f f o r m i c a c i d w i t h 1.0 ml o f 30% h y d r o g e n p e r o x i d e ) . The m i x t u r e was a l l o w e d t o r e a c t f o r k h r a t -10 C and was t h e n l y o p h i l i z e d . ( c ) C a r b o x a m i d o m e t h y l a t e d F e r r e d o x i n (CAM-Fd) T C A - F d was d i s s o l v e d t o a c o n c e n t r a t i o n o f 1-2 mg/ml i n d i s t i l l e d w a t e r a d j u s t e d t o pH 8.0 w i t h d i l u t e NaOH. R e d u c t i o n o f c y s t e i n y l r e s i d u e s was e n s u r e d by a d d i n g an 8 0 - f o l d m o l a r e x c e s s o f 2 - m e r c a p t o e t h a n o l f o l l o w e d by i n c u b a t i o n a t kj C f o r k h r . The p r o t e i n was p r e c i p i t a t e d by g r a d u a l l y a c i d i f y i n g t h e s o l u t i o n w i t h k N HC1. The p r e c i p i t a t e was c e n t r i f u g e d , r e -s u s p e n d e d i n w a t e r , and an e q u a l amount (w/w) o f i o d o a c e t a m i d e was a d d e d . T h e pH was t h e n a d j u s t e d t o 8.0 w i t h d i l u t e NaOH and a l k y l a t i o n was a l l o w e d ' t o o c c u r f o r 10 m i n a t room t e m p e r a -t u r e . The pH w a s , a g a i n l o w e r e d w i t h k N HC1 and t h e p r e c i p i t a t e d p r o t e i n was c o l l e c t e d by c e n t r i f u g a t i o n and w a s h e d o n c e w i t h d i s t i l l e d w a t e r b e f o r e b e i n g d i s s o l v e d i n t h e a p p r o p r i a t e b u f f e r . i m m u n i z a t i o n P r o c e d u r e . P u r i f i e d C_. p a s t e u r i a n u m f e r r e d o x i n u s e d f o r p r o d u c t i o n o f a n t i -s e r a was p u r c h a s e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o . A n t i s e r a w e r e r a i s e d i n r a b b i t s t o n a t i v e f e r r e d o x i n and i t s p e r f o r m i c a c i d o x i d i z e d d e r i v a t i v e . F i v e mg o f a n t i g e n w e r e d i s s o l v e d i n 0 \ 5 ml p h y s i o l g i c a l s a l i n e and e m u l s i f i e d i n an e q u a l v o l u m e o f c o m p l e t e F r e u n d ' s a d j u v a n t ( D i f c o ) . R a b b i t s w e r e a n a e s t h e t i z e d w i t h an i n t r a v e n o u s i n j e c t i o n o f 1 . 0 ml n e m b u t a l ( H y l a n d C o . ) and 0 . 2 ml o f t h e a d j u v a n t p r e p a r a t i o n was i n j e c t e d i n t o a f o o t pad o f e a c h e x t r e m i t y a n d 0 . 2 ml was i n j e c t e d i n t r a m u s c u l a r l y i n t h e l e f t h i n d l e g . The f o l l o w i n g d a y , 1 . 0 mg o f a l u m p r e c i p i t a t e d a n t i g e n , p r e p a r e d a c c o r d i n g t o t h e method o f K a b a t and M a y e r ( . 1 9 6 1 ) , was i n j e c t e d i n t r a v e n o u s l y i n an e a r v e i n . A f t e r k w e e k s , e a r b l e e d s w e r e p e r f o r m e d w e e k l y and t h e s e r u m was s t o r e d i n 50 ml b a t c h e s a f t e r h e a t i n g a t 55 C f o r 30 m i n t o d e s t r o y c o m p l e m e n t a c t i v i t y . T i t e r s w e r e f o u n d t o be s t a b l e o v e r a s i x - m o n t h p e r i o d , a t w h i c h t i m e a b o o s t e r i n j e c t i o n o f 1 mg o f a n t i g e n i n 0 . 5 ml c o m p l e t e F r e u n d ' s a d j u v a n t was a d m i n i s t e r e d i n t h e l e f t h i n d l e g . M e r t h i o l a t e a t a c o n c e n t r a t i o n o f 1 : 1 0 , 0 0 0 was a d d e d t o t h e s e r a a s a p r e s e r v a t i v e . V I . C o m p l e m e n t F i x a t i o n The c o m p l e m e n t f i x a t i o n r e a c t i o n w a s , u s e d f o r i n i t i a l s t u d i e s on t h e a n t i g e n i c p r o p e r t i e s o f f e r r e d o x i n and i t s c y s t e i n e - m o d i f i e d d e r i v a t i v e s . F r e e z e - d r i e d g u i n e a p i g s e r u m ( 3 . 0 ml q u a n t i t i e s ) p u r c h a s e d f r o m H y l a n d C o . was u s e d a s t h e s o u r c e o f c o m p l e m e n t . T h e s e r u m was r e c o n s t i t u t e d f o r e a c h t e s t i n t h e d i l u e n t p r o v i d e d and f u r t h e r d i l u t i o n s w e r e made i n v e r o n a l b u f f e r e d s a l i n e , b e t w e e n 1 : 2 5 a n d 1 : 2 5 0 , b e t w e e n 1 : 5 0 and 1 : 5 0 0 o r b e t w e e n 1 : 7 5 and 1 : 7 5 0 , d e p e n d i n g on t h e b a t c h o f g u i n e a p i g s e r u m u s e d . The v e r o n a l b u f f e r e d s a l i n e u s e d f o r a l l d i l u t i o n s o f a n t i g e n , a n t i s e r u m , c o m p l e m e n t a n d h a p t e n was p r e p a r e d a c c o r d i n g t o t h e m e t h o d o f B r o o k s b y ( 1 9 5 2 ) . T h e c o m p l e m e n t f i x a t i o n t i t e r s o f t h e two a n t i s e r a w e r e d e t e r m i n e d by u s i n g s e r i a l d i l u t i o n s o f c o m p l e m e n t b e t w e e n t h e v a l u e s g i v e n a b o v e , d o u b l i n g d i l u t i o n s o f a n t i s e r a b e t w e e n 1 : 1 0 and 1 : 3 2 0 and one d i l u t i o n o f a n t i g e n ( 0 . 5 u g ) . T h e a n t i g e n , a n t i s e r u m and c o m p l e m e n t w e r e a l l u s e d i n 1 . 0 ml q u a n t i t i e s p e r t u b e . A s i m i l a r t e s t t o c h e c k t h e a n t i c o m p l e m e n t a r y a c t i v i t y o f t h e a n t i s e r u m was s e t up s i m u l t a n e o u s l y u s i n g 1 . 0 ml o f v e r o n a l b u f f e r e d s a l i n e i n s t e a d o f a n t i g e n . The t u b e s w e r e m i x e d by s h a k i n g and i n c u b a t e d o v e r n i g h t a t h C . A 2% s u s p e n s i o n o f t w i c e - w a s h e d s h e e p e r y t h r o c y t e s i n v e r o n a l b u f f e r e d s a l i n e was s e n s i t i z e d by m i x i n g them w i t h an e q u a l v o l u m e o f 1 : 5 0 d i l u t i o n o f a s t a n d a r d s o l u t i o n o f s h e e p h e m o l y s i n ( D i f c o ) ; f o r 10 m i n . T h i s p r e p a r a t i o n was a d d e d i n 1 . 0 ml a m o u n t s t o t h e t e s t s e r i e s w h i c h w e r e t h e n i n c u b a t e d a t 37 C f o r 30 m i n i n a w a t e r b a t h . T u b e s w e r e s h a k e n o n c e a t 15 m i n . F o l l o w i n g t h e 30 m i n i n c u b a t i o n , u n h e m o l y z e d r e d c e l l s and c e l l d e b r i s w e r e s e d i m e n t e d by c e n t r i f u g a t i o n a t J 8 0 0 x g_ f o r 10 m i n , and t h e s u p e r n a t a n t s w e r e r e a d on a K l e t t c o l o r i m e t e r w i t h a g r e e n f i l t e r . End p o i n t s w e r e c a l c u l a t e d a s 50% h e m o l y s i s on a p r o b i t p l o t o f a c t u a l p e r c e n t a g e s o f t h e K l e t t r e a d i n g s a c c o r d i n g t o t h e m e t h o d d e s c r i b e d by W r i g h t ( 1 9 6 3 ) . T h e t i t e r o f t h e n a t i v e f e r r e d o x i n a n t i -s e r u m was c a l c u l a t e d t o be 1:h0 and t h a t o f t h e o x i d i z e d f e r r e d o x i n a n t i s e r u m 1 : 2 0 0 . No a n t i c o m p l e m e n t a r y a c t i v i t y c o u l d be d e t e c t e d a t t h e s e d i l u t i o n s o f a n t i s e r u m . The d e g r e e o f c r o s s - r e a c t i o n b e t w e e n t h e two a n t i s e r a and t h e f o u r a n t i g e n p r e p a r a t i o n s ( n a t i v e F d , T C A - F d , OFd and CAM-Fd) was d e t e r -m i n e d u s i n g t h e c o m p l e m e n t f i x a t i o n r e a c t i o n . In t h i s c a s e , a c o n s t a n t d i l u t i o n o f a n t i s e r u m was u s e d t h r o u g h t o u t (1:k0 f o r n a t i v e Fd a n t i -s e r u m a n d 1:200 f o r o x i d i z e d f e r r e d o x i n a n t i s e r u m , a s p r e v i o u s l y d e t e r m i n e d ) , m i x e d w i t h d o u b l i n g d i l u t i o n s o f e a c h a n t i g e n r a n g i n g f r o m 12 y g t o 0.25 y g a t e a c h o f t h e t e n d i l u t i o n s o f c o m p l e m e n t , e a c h i n a t o t a l v o l u m e o f 1.0 m l . The i n c u b a t i o n t i m e s , t e m p e r a t u r e s and a s s a y m e t h o d w e r e i d e n t i c a l w i t h t h o s e u s e d f o r d e t e r m i n i n g t h e t i t e r s o f t h e a n t i s e r a . E n d p o i n t s w e r e c a l c u l a t e d a s t h e 50% h e m o l y s i s p o i n t f r o m p r o b i t p l o t s a s d e s c r i b e d a b o v e . V I I . Q u a n t i t a t i v e P r e c i p i t i n R e a c t i o n The d e g r e e o f c r o s s - r e a c t i o n was a l s o d e t e r m i n e d u s i n g t h e q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n . The a n t i s e r u m t o OFd a t a d i l u t i o n o f 1:4 was d i s p e n s e d i n 0.5 ml q u a n t i t i e s t o a s e r i e s o f t u b e s . T h e v a r i o u s a n t i g e n p r e p a r a t i o n s , a l s o i n a v o l u m e o f 0.5 m l , w e r e a d d e d i n a m o u n t s v a r y i n g f r o m 0.25 t o 25 y g • E a c h t e s t was p e r f o r m e d i n t r i p l i c a t e . A n e g a t i v e c o n t r o l was s e t up u s i n g 0.5 ml o f s a l i n e i n s t e a d o f a n t i g e n . T h e d i l u e n t u s e d t h r o u g h o u t was 0.3% s a l i n e . T u b e s w e r e m i x e d and i n c u b a t e d f o r 1 h r i n a 37 C w a t e r b a t h a n d t h e n o v e r n i g h t a t k C . T h e f o l l o w i n g d a y , t h e p r e c i p i t a t e d w e r e s e d i -m e n t e d i n a c l i n i c a l c e n t r i f u g e s a n d w a s h e d t w i c e w i t h 0.3% s a l i n e . The w a s h e d p r e c i p i t a t e s w e r e d i s s o l v e d i n 0 . 1 N NaOH and a b s o r b a n c e s a t 280 m u , 1 . 0 cm l i g h t p a t h , w e r e r e a d on a Beckman DBG s p e c t r o -p h o t o m e t e r . V I I I . P e p t f d e S y n t h e s i s The C O O H - t e r m i n a l o c t a p e p t i d e o f C_. p a s t e u r i a n u m f e r r e d o x i n was s y n t h e s i z e d u s i n g t h e s o l i d p h a s e m e t h o d o f M e r r i f i e l d (.1964) a s d e s c r i b e d by S t e w a r t and Young ( 1 9 6 9 ) - T h e c h l o r o m e t h y l a t e d p o l y - r s t y r e n e - d i v i n y l b e n z e n e c o p o l y m e r r e s i n was p u r c h a s e d f r o m B i o r a d C o . , and t h e p r o t e c t e d t - b u t y l o x y c a r b o n y l ( t - b o c ) a m i n o a c i d s w e r e p u r c h a s e d f r o m Mann R e s e a r c h B i o c h e m i c a l s and S i g m a C h e m i c a l C o . One g r a m o f r e s i n was u s e d f o r t h e s y n t h e s i s p r o c e d u r e , and p r o t e c t e d a m i n o a c i d s w e r e a d d e d In a 3-5 f o l d m o l a r e x c e s s o f t h e amount o f t h e i n i t i a l a m i n o a c i d a t t a c h e d , e x c e p t f o r a s p a r a g i n e , w h i c h was a d d e d i n a 5 - f o l d ' m o l a r e x c e s s . The p e p t i d e was c l e a v e d f r o m t h e r e s i n by b u b b l i n g a n -h y d r o u s HBr ( M a t h e s o n C o . ) t h r o u g h a s u s p e n s i o n o f t h e r e s i n i n 1 0 . 0 ml o f a n h y d r o u s t r i f 1 u o r o a c e t i c a c i d f o r 90 m i n ; t h i s p r o c e d u r e a l s o . removed t h e p r o t e c t i n g 0 - b e n z y l g r o u p f r o m t h e g l u t a m i c a c i d r e s i d u e . T r i f l u o r o a c e t i c a c i d w a s . removed by f l a s h e v a p o r a t i o n and l y o p h i l i -z a t i o n . The p e p t i d e was p u r i f i e d f u r t h e r by c o l u m n c h r o m a t o g r a p h y on Dowex A g - 1 - X 2 r e s i n ( 2 0 0 - 4 0 0 m e s h - B i o r a ' d C o . ) , a c c o r d i n g t o t h e m e t h o d o f K o n i g s b e r g and H i l l ( 1 9 6 2 ) . The p r e p a r e d r e s i n was p o u r e d i n t o a 1.5 cm d i a m e t e r c o l u m n t o a h e i g h t o f 27 cm a n d e q u i 1 " i b r a t e d w i t h . N - e t h y l m o r p h o l i n e - a c e t a t e b u f f e r ( 0 . 2 M i n a c e t a t e ) a t pH 8.6. The p e p t i d e was a p p l i e d t o t h e c o l u m n i n a v o l u m e o f 2 . 0 ml o f t h i s b u f f e r . T h e c o l u m n was d e v e l o p e d w i t h a g r a d i e n t : t h e m i x i n g c h a m b e r c o n t a i n e d 50 ml o f t h e pH 8 . 6 b u f f e r a n d t h e r e s e r v o i r c o n t a i n e d 50 ml o f 1 . 0 M p y r i d i n e - a c e t a t e b u f f e r a t pH 6 . 5 6 . A f t e r t h e g r a d i e n t was c o m p l e t e d , 50 ml o f 2 M p y r i d i n e - a c e t a t e b u f f e r a t pH 4 . 0 5 was r u n t h r o u g h t h e c o l u m n . A f l o w r a t e o f 20 m l / h r was m a i n t a i n e d and 4 . 0 ml s a m p l e s w e r e c o l l e c t e d on an LKB f r a c t i o n c o l l e c t o r . A 0 . 2 ml a l i q u o t was t a k e n f r o m e v e r y o t h e r f r a c t i o n f o r d e t e r m i n a t i o n o f p e p t i d e c o n t e n t by t h e q u a n t i t a t i v e n i n h y d r i n m e t h o d o f H i r s et^ a_l_ ( 1 9 6 6 ) a f t e r a l k a l i n e h y d r o l y s i s was c a r r i e d o u t a c c o r d -i n g t o t h e m e t h o d o f S t e w a r t a n d Young ( 1 9 6 9 ) . The p e p t i d e - c o n t a i n i n g f r a c t i o n s w e r e p o o l e d and l y o p h i l i z e d . The d r i e d p e p t i d e s a m p l e s w e r e d i s s o l v e d i n 2 . 0 ml o f d i s t i l l e d w a t e r . The p e p t i d e s w e r e c h e c k e d f o r p u r i t y by h i g h V o l t a g e e l e c t r o p h o r e s i s and t h e i r c o m p -o s i t i o n s d e t e r m i n e d u s i n g t h e a m i n o a c i d a n a l y z e r . IX . H a p t e n I n h i b i t i o n S t u d i e s The h a p t e n i c a c t i v i t y o f . t h e s y n t h e s i z e d p e p t i d e was t e s t e d by i n h i b i t i o n o f c o m p l e m e n t f i x a t i o n and b y i n h i b i t i o n o f p r e c i p i t a t i o n . B o t h t e s t s w e r e c a r r i e d o u t b a s i c a l l y a s d e s c r i b e d by G e r w t n g and Thompson ( 1 9 6 9 ) . F o r i n h i b i t i o n o f c o m p l e m e n t f i x a t i o n , n a t i v e Fd and OFd a n t i s e r a a t . 1 : 4 0 a n d 1 : 2 0 0 d i l u t i o n s ' r e s p e c t i v e l y w e r e m i x e d i n 1.0 ml volumes w i t h amounts o f p e p t i d e v a r y i n g between 0.25 and 25 ug i n 0.10 ml q u a n t i t i e s , each o v e r a range o f 10 complement d i l u t i o n s . A p o s i t i v e c o n t r o l was s e t up u s i n g 0.1 ml o f v e r o n a l b u f f e r e d s a l i n e i n s t e a d o f p e p t i d e f o r one s e r i e s o f t u b e s . The tubes were i n c u b a t e d f o r 1 hr i n a 37 C water bath and then o v e r n i g h t a t k C. The f o l l o w i n g day, 1.0 ml o f the homologous a n t i g e n (0.5 ug) and 1.0 ml o f t h e a p p r o p r i a t e d i l u t i o n o f complement was added t o each t u b e . I n c u b a t i o n was c a r r i e d out o v e r n i g h t a t k C. On the t h i r d day t h e h e m o l y s i n -e r y t h r o c y t e i n d i c a t i n g s ystem was added and h e m o l y s i s d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d . The amount o f i n h i b i t i o n was c a l c u l a t e d as the d i f f e r e n c e i n p e r c e n t a g e h e m o l y s i s between the tubes i n the t e s t s e r i e s and t h o s e o f t h e p o s i t i v e c o n t r o l c o n t a i n i n g t h e same d i l u t i o n o f complement when compared w i t h a no a n t i g e n c o n t r o l , w h i c h was t a k e n a t 100% h e m o l y s i s . The tubes i n the p o s i t i v e c o n t r o l on e i t h e r s i d e o f the 50% h e m o l y s i s p o i n t were chosen f o r c a l c u l a t i o n . To d e t e r m i n e t h e degree o f i n h i b i t i o n o f p r e c i p i t a t i o n caused by t h e p e p t i d e , a m o d i f i c a t i o n o f the p r o c e d u r e d e s c r i b e d by GerwTng and Thompson (19&9) was used. V a r y i n g amounts o f hapten between 500 and 5 u g , i n a volume o f 0.1 ml were mixed i n t r i p l i c a t e w i t h a c o n s t a n t amount o f a n t i s e r u m i n 0.5 ml a t a 1:4 d i l u t i o n . Tubes were i n c u b a t e d f o r 1 hr i n a 37 C w a t e r b a t h and then a n t i g e n a t a c o n c e n t r a t i o n p r e -v i o u s l y d e t e r m i n e d t o be s l i g h t l y i n e x c e s s o f optimum was added i n 0.5 ml q u a n t i t i e s . I n c u b a t i o n was c o n t i n u e d f o r a n o t h e r hour a t 37 C. The p r e c i p i t a t e s formed were s e d i m e n t e d , washed once w i t h s a l i n e and 31 •dissolved in 1.0 ml of 0.1 N NaOH and the absorbances read at 280/mu, 1.0 cm l i g h t p a t h , on a Beckman DBG spectrophotometer . X. E q u i l i b r i u m D i a l y s i s (a) P u r i f i c a t i o n of Ant ibody . S p e c i f i c a n t i b o d i e s d i r e c t e d a g a i n s t n a t i v e f e r r e d o x i n and i t s per fo rmic a c i d o x i d i z e d d e r i v a t i v e were p u r i f i e d from t h e i r r e s p e c t i v e a n t i s e r a us ing a s p e c i f i c immunoadsorbent. An OFd immunoadsorbent was used to o b t a i n o a n t i b o d i e s from each serum. The method of p r e p a r a t i o n of the immunoadsorbent and p u r i f i c a t i o n of ant ibody was b a s i c a l l y that of Robbins et "al 0 9 6 7 ) . Seventy -f i v e mg of OFd were" c o v a l e n t l y bonded to 1 .0 g of bromoacetyl c e l l u l o s e (prepared by r e a c t i o n of bromoacetic a c i d and bromo-a c e t y l bromide w i t h 1.0 g of Whatman powdered eel 1ulose) In 0.15 M c i t r a t e - p h o s p h a t e b u f f e r pH 4.6. Before a d s o r p t i o n , 75 ml of each serum was c l a r i f i e d by c e n t r i f u g a t i o n at 20,000 x g_ f o r 1 h r , and sedimented d e b r i s and f l o a t i n g l i p i d m a t e r i a l were removed. The c e l l u l o s e conjugate was then d i s p e r s e d ' i n the serum, arid the suspension was s t i r r e d at 4 C f o r 2 h r . Then the adsorbed conjugate was removed by c e n t r i f u g a t i o n at 20,000 x g_ f o r 20 m i n , arid a l l supernatant serum was dra ined o f f . The conjugate was washed w i t h 0.15 M NaCl and r e c e n t r i f u g e d u n t i l the absorbance of the supernate at 280 mu was less than 0.08. The ant ibody w a s e l u t e d by s t i r r i n g t h e c o m p l e x i n 1 0 - 1 5 ml o f 0 . 1 M a c e t i c a c i d (pH 2 . 8 ) f o r 1 h r a t 37 C. The a d s o r b e n t was removed by c e n t r i f u g a t i o n a t 2 0 , 0 0 0 x g f o r 20 m i n , a n d t h e s u p e r n a t a n t . f l u i d c o n t a i n i n g t h e a n t i b o d y ' was d i a l y z e d a g a i n s t 3 5 0 - 7 0 0 v o l u m e s o f 0 . 1 M N a C 1 - 0 . 0 1 M T r i s -HC1 b u f f e r pH 7 . 0 . The p u r i f i e d a n t i b o d y was q u a n t i t a t e d by t h e L o w r y m e t h o d a s d e s c r i b e d by K a b a t and M a y e r ( 1 9 6 1 ) , and t h e t i t e r o f t h e s o l u t i o n was d e t e r m i n e d by c o m p l e m e n t f i x a t i o n . P u r i f i e d a n t i b o d y was s t o r e d a t k C i n t h e T r i s b u f f e r d e s c r i b e d a b o v e t o w h i c h 60 y g m e r t h i o l a t e ml had been a d d e d . A f t e r w a s h i n g o n c e w i t h 0 . 1 M a c e t i c a c i d , t h e i m m u n o a d s o r b e n t w a s . s t o r e d a t k C s u s p e n d e d i n 0 . 1 5 M p h o s p h a t e b u f f e r pH 7 . 6 . (b) P u r i f i c a t i o n o f N o n - s p e c i f i c Gamma G l o b u l i n Gamma g l o b u l i n , f o r u s e a s a c o n t r o l , was p u r i f i e d f r o m n o n -s p e c i f i c r a b b i t s e r u m by p r e c i p i t a t i o n w i t h 50% ammonium s u l p h a t e , d i a l y s i s , and s u b s e q u e n t c o l u m n c h r o m a t o g r a p h y on DEAE c e l l u l o s e w i t h 0 . 0 5 M p h o s p h a t e b u f f e r pH 7 - 5 a c c o r d i n g t o t h e m e t h o d o f F u j i o et^ aj_ ( 1 9 6 8 a ) . T h e gamma g l o b u l i n was s t o r e d a s a l y o p h i l i z e d p o w d e r . F o r u s e i n e q u i l i b r i u m d i a l y s i s , s m a l l a m o u n t s o f t h e powder w e r e d i s s o l v e d i n PBS ( p h o s p h a t e b u f f e r e d s a l i n e : 0 . 0 2 M p h o s p h a t e b u f f e r pH 7 . 0 , 0 . 1 5 % N a C l and 0 . 0 2 % s o d i u m a z i d e ) and e x h a u s t i v e l y d i a l y z e d a t k C a g a i n s t t h e same b u f f e r . The p r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by t h e L o w r y m e t h o d . ( c ) (1- C ) - A c e t y l a t i o n o f t h e P e p t i d e 1 4 T h e p r e p a r a t i o n o f t h e ( 1 - C ) - a c e t y l a t e d p e p t i d e was c a r r i e d o u t a c c o r d i n g t o t h e m e t h o d o f F u j i o et_ a_l_ (1968b). The ( 1 - ^ C ) -a c e t i c a n h y d r i d e (5mCi/mM) was p u r c h a s e d f r o m New E n g l a n d N u c l e a r C o r p . i n 0.10 C i b r e a k s e a l t u b e s . T h e t u b e was f r o z e n i n an a c e t o n e - d r y i c e b a t h and 1 . 5 ml o f a n h y d r o u s b e n z e n e was p l a c e d o v e r t h e vacuum s e a l . Once t h e s e a l w a s . b r o k e n , t h e b e n z e n e was d r a w n i n t o t h e t u b e , d i s s o l v i n g t h e l a b e l l e d a n h y d r i d e . T h e b e n z e n e s o l u t i o n was warmed s l i g h t l y and d r a w n o u t o f t h e t u b e w i t h a s y r i n g e a n d t r a n s f e r r e d t o a s m a l l s c r e w - c a p p e d v i a l . F o u r t e e n u m o l e s o f t h e p e p t i d e w e r e d i s s o l v e d i n 1 . 5 ml o f 1 . 0 M s o d i u m a c e t a t e a d j u s t e d t o pH 8 . 0 w i t h NaOH. A 2 0 - f o l d m o l a r e x c e s s o f l a b e l l e d a c e t i c a n h y d r i d e i n b e n z e n e was l a y e r e d on t o p o f t h e a q u e o u s p e p t i d e s o l u t i o n i n a s m a l l s c r e w - c a p p e d v i a l . A f t e r /6 h r a t 4 C , t h e a q u e o u s p h a s e w a s : w i t h d r a w n and p a s s e d t h r o u g h a 2 . 5 cm x 40 cm c o l u m n o f S e p h a d e x G-15 ( P h a r m a c i a ) e q u i l i b r a t e d w i t h 50% a c e t i c a c i d . The c o l u m n was e l u t e d w i t h t h e same s o l v e n t a t 25 C . F r a c t i o n s o f 4 . 5 ml w e r e c o l l e c t e d and 0 . 1 0 ml f r o m e v e r y • s e c o n d t u b e w e r e m i x e d w i t h 1 0 . 0 ml o f s c i n t f a l l a t i o n f l u i d and t h e r a d i o a c t i v i t y m e a s u r e d on a N u c l e a r C h i c a g o S c i n t i l l a t i o n C o u n t e r M o d e l 7 5 0 . T h e s c i n t i l l a t i o n f l u i d was p r e p a r e d a s f o l l o w s : 4 2 . 0 ml L i q u i f l u o r (New E n g l a n d N u c l e a r C o r p . ) and "383.2 ml m e t h a n o l d i s s o l v e d T n 547.8 ml t o l u e n e . The p e p t f d e - c o n t a i n i n g f r a c t i o n s w e r e p o o l e d and t a k e n t o d r y n e s s by f l a s h e v a p o r a t i o n , u s i n g a s p e c i a l d r y - i c e a c e t o n e t r a p . T h e p e p t i d e was r e d i s s o l v e d iri 2.0 ml d i s t i l l e d w a t e r , l y o p h i l i z e d and t h e n d i s s o l v e d i n P B S . D u p l i c a t e 0.01 ml s a m p l e s w e r e m i x e d w i t h 10.0 ml o f t h e s c i n t i l l a t i o n f l u i d and t h e r a d i o -a c t i v i t y was m e a s u r e d t o d e t e r m i n e t h e amount o f l a b e l p r e s e n t on t h e p e p t i d e . A s m a l l amount o f p e p t i d e was a l s o t a k e n f o r a m i n o a c i d a n a l y s i s s o t h a t s p e c i f i c a c t i v i t y c o u l d be e s t a b l i s h e d . (d) D i a l y s i s P r o c e d u r e E q u i l i b r i u m d i a l y s i s e x p e r i m e n t s w e r e s e t up u s i n g p e n c i l d i a l y s i s t u b i n g ( V i s k i n g - U n i o n Carbide-1 Cm) and s m a l l s c r e w - c a p p e d v i a l s . P h o s p h a t e - b u f f e r e d s a l i n e was u s e d a s d i l u e n t t h r o u g h o u t . P u r i f i e d a n t i s e r a t o b o t h n a t i v e f e r r e d o x i n and o x i d i z e d f e r r e -d o x i n , c o n t a i n i n g b e t w e e n 300 t o hOO u g p r o t e i n / m l , w e r e a d d e d t o i n d i v i d u a l d i a l y s i s t u b e s i n 1.0 ml q u a n t i t i e s . C o n t r o l s w e r e s e t up u s i n g 1.0 ml v o l u m e s o f PBS and n o n - s p e c i f i c gamma g l o b u l i n , a l s o i n i n d i v i d u a l d i a l y s i s t u b e s ; T h e d i a l y s i s s a c s w e r e i m m e r s e d i n d i v i d u a l l y in'10.0 ml o f t h e l a b e l l e d p e p t i d e s o l u t i o n , a t c o n c e n t r a t i o n s o f 0.002 a n d 0.001 u m o l e s / m l . The v i a l s w e r e p l a c e d i n a s h a k e r a t k C , and d i a l y s i s was a l l o w e d t o c o n t i n u e f o r k d a y s , a t w h i c h t i m e e q u i l i b r i u m was r e a c h e d , a s e s t a b l i s h e d by m e a s u r e m e n t o f t h e PBS c o n t r o l . T h e n 0.10 ml s a m p l e s f r o m i n s i d e and o u t s i d e e a c h d i a l y s i s membrane w e r e t a k e n arid t h e r a d i o a c t i v i t y was m e a s u r e d . D u p l i c a t e c o u n t s p e r m i n u t e , w e r e e s t i m a t e d f r o m 10 m i n c o u n t s o f e a c h s a m p l e . RESULTS AND DISCUSSION I . P r e p a r a t i o n o f F e r r e d o x i n The a n t i g e n i c i t y o f f e r r e d o x i n f r o m C l o s t r i d i u m p a s t e u r i a n u m was s t u d i e d i n an a t t e m p t t o d e t e r m i n e t h e a n t i g e n i c b i n d i n g s i t e s o f t h e p r o t e i n . T h i s m o l e c u l e was c o n s i d e r e d a good s u b j e c t f o r s u c h a s t u d y s i n c e t h e a m i n o a c i d s e q u e n c e s o f f e r r e d o x i n s f r o m s e v e r a l r e l a t e d s p e c i e s w h i c h h a v e b e e n p u b l i s h e d ( T s u n o d a and Y a s u n o b u , 1968),. show e x t e n s i v e s e q u e n c e h o m o l o g y , and b e c a u s e i t i s a s m a l l p r o t e i n w h i c h s h o u l d p o s s e s s r e l a t i v e l y f e w a n t i g e n i c d e t e r m i n a n t s . F o r u s e i n i m m u n o l o g i c a l t e s t i n g o f t h e a n t i s e r a , f e r r e d o x i n was p u r i f i e d f r o m c e l l p r e p a r a t i o n s o f C_. p a s t e u r i a n u m g r o w n i n t h e l a b o r a t o r y . F o u r 30 l i t r e c u l t u r e s w e r e g r o w n i n a c o m p l e t e l y s y n t h e t i c medium and h a r v e s t e d by c o n t i n u o u s f l o w c e n t r i f u g a t i o n . The y i e l d o f c e l l p a s t e f o r t h e f o u r ' b a t c h e s o f c u l t u r e U s g i v e n i n T a b l e 1. U s i n g t h e c o n t i n u o u s f l o w m e t h o d o f c e n t r i f u g a t i o n as d e s c r i b e d i n t h e M e t h o d s , a c o m p l e t e c u l t u r e c o u l d be h a r v e s t e d I'ri f o u r h o u r s w i t h o u t s t o p p i n g t h e c e n t r i f u g e . The d e c r e a s e i n o p t i c a l d e n s i t y a t 650 m y i b e t w e e n t h e c u l t u r e and t h e e f f l u e n t w a s , c a l c u l a t e d 5 t o be 99-9%- • F e r r e d o x i n was p u r i f i e d f r o m e a c h b a t c h o f c e l l s e p a r a t e -l y . The p u r i f i c a t i o n p r o c e d u r e t a k e s a d v a n t a g e o f t h e f a c t t h a t f e r r e d o x i n i s s o l u b l e i n a 50% a q u e o u s s o l u t i o n o f a c e t o n e w h i l e 99% o f t h e r e m a i n d e r o f t h e c e l l u l a r c o n s t i t u e n t s a r e n o t ; and o f t h e f a c t Batch No. Wt. C e l l Paste Wt. Ferredoxin 390 mu/280 mu r a t i o 1 301 g. 56 mg. 0.?8 2 432 g. 62 mg. 0.73 3 205 g. 35 mg. 0.71* k 350 g. 42 mg. 0.72 Table 1. Y i e l d of c e l l paste and p u r i f i e d ferredoxin with the 390 mu/280 mu r a t i o s found f o r the four batches p u r i f i e d . t h a t f e r r e d o x i n i s e x t r e m e l y e l e c t r o n e g a t i v e ( p i = 3.7, L o v e n b e r g , et_ a_j_, 1963) and w i l l a d s o r b t o an a n i o n e x c h a n g e r e s i n u n d e r c o n d i t i o n s w h i c h a l l o w m o s t . o f t h e r e m a i n i n g c o n t a m i n a t i n g m a t e r i a l t o be w a s h e d o u t . F i n a l p u r i f i c a t i o n was a c h i e v e d by ammonium s u l p h a t e f r a c t i o n -a t i o n . The f i n a l p r o d u c t was d a r k brown a n d r e a d i l y s o l u b l e i n d i s t i l l e d w a t e r o r b u f f e r . T h e y i e l d o f p u r i f i e d f e r r e d o x i n f r o m e a c h o f t h e f o u r b a t c h c u l t u r e s i s a l s o shown i n T a b l e 1. T h r e e c r i t e r i a w e r e u s e d t o e s t a b l i s h t h e d e g r e e o f p u r i t y o f t h e f i n a l p r e p a r a t i o n s : ( i ) The o p t i c a l a b s o r p t i o n s p e c t r u m was d e t e r m i n e d f o r e a c h s a m p l e . F e r r e d o x i n s h o u l d show a b s o r p t i o n maxima a t 390 my and 280 m y , and t h e r a t i o b e t w e e n t h e two i s a n i n d i c a t i o n o f t h e r e l a t i v e p u r i t y o f t h e p r e p a r a t i o n . F i g u r e 4 shows a r e p r e s e n t a t i v e a b -s o r p t i o n s p e c t r u m o f a p u r i f i e d p r e p a r a t i o n w h i c h , e x h i b i t s t h e t y p i c a l m a x i m a . T h e 390 my/280 my r a t i o s o f t h e f o u r p r e p a r a t i o n s a r e a l s o l i s t e d i n T a b l e 1. T h e y v a r y f r o m 0.72 t o 0.78, w h i c h a r e i n f a i r a g r e e m e n t w i t h t h e r e p o r t e d v a l u e o f B u c h a n a n et^ a_l_ (1963) o f 0.80 and w i t h t h a t o f t h e c o m m e r c i a l p r e p a r a t i o n o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p . , w i t h a r a t i o o f 0.75 ( i i ) O n l y a s i n g l e , n e g a t i v e l y c h a r g e d ' n i n h y d r i n - p o s i t i v e s p o t c o u l d be d e t e c t e d i n e a c h s a m p l e a f t e r h i g h v o l t a g e e l e c t r o -p h o r e s i s . ( i i i ) The a m i n o a c i d c o m p o s i t i o n o f e a c h s a m p l e was d e t e r m i n e d . T h e c o m p o s i t i o n o f a r e p r e s e n t a t i v e s a m p l e i s l i s t e d i i i T a b l e 2, a n d , f o r c o m p a r i s o n , t h e c o m p o s i t i o n r e p o r t e d by M o r t e n s o n (1964) i s a l s o g i v e n . T h e f a c t t h a t l e u c i n e , h i s t i d i n e and a r g i n i n e 38 250 300 350 WAVELENGTH mjj 400 F i g . k. S p e c t r u m a n a l y s i s o f C . p a s t e u r i a n u m f e r r e d o x i n . GM/100 GM Protein Amino Acid Fd* Fd TCA-Fd O-Fd CAE-Fd Lysine 3.0 2.8 Aspartic a c i d 17-0 15.3 Threonine 1.3 1.9 Serine k.9 5.1 Glutamic a c i d 10.6 7.6 Proline 6.3 6.5 Glycine k.2 9.5 Alanine 9.8 13. h Cysteine 5.3 8.7 Cysteic acid Carboxamido-methylcysteine Valine . 10.0 12.0 Isoleucine 9.8 8.7 Tyrosine 2.8 1.6 Phenylalanine 2.6 2.7 2.8 2.9 ...3.0 16.8 17.1 16.0 2.U5 1.7 2.6 5.8 2.7 7.1 11.2 9.0 8.2 6.3 7.7 5.U 10.3 8.3 8.2 13.2 1U.2 13.6 6.8 12.7 9.5 11.7 12.5 11.3 9.k 8.3 9.U 0.8k 0.5 2.0 1.9 2.2 2.7 I Table 2. The amino.acid compositions of the various antigenic preparations used i n these studies. * Amino a c i d composition f o r native Fd reported by Mortenson (1964). c o u l d n o t be d e t e c t e d i n a m o u n t s e x c e e d i n g 0 . 1 g p e r 100 g p r o t e i n i n d i c a t e d t h a t t h e p r e p a r a t i o n s w e r e v i r t u a l l y f r e e o f p e p t i d e c o n t a m i n a n t s . I I . A n t i s e r a C a r p e n t e r ( 1 9 6 5 ) r e p o r t s t h a t t h e a v e r a g e m o l e c u l a r w e i g h t f o r a n t i g e n i c s u b s t a n c e s i s 1 0 , 0 0 0 ; w i t h t h e s m a l l e s t a n t i g e n s f o u n d t o d a t e b e i n g a s y n t h e t i c h o m o p o l y m e r o f g l u t a m i c a c i d w i t h a m o l e c u l a r w e i g h t o f 4100 ( S e l a , 1 9 6 6 ) , and a p h e n y l i s o c y a n a t e o f c l u p e i n o f m o l e c u l a r w e i g h t 5 0 0 0 ( C a r p e n t e r , . 1 9 6 5 ) . F e r r e d o x i n , w i t h a m o l e c u l a r w e i g h t o f 5 5 0 0 a p p r o a c h e s t h i s l i m i t ' o f s i z e f o r a n t i g e n i c i t y . T h e r e -f o r e , t h e f i r s t p r o b l e m p r e s e n t e d was t o d e t e r m i n e i f f e r r e d o x i n c o u l d e v o k e an a n t i b o d y r e s p o n s e i n r a b b i t s . B o t h n a t i v e f e r r e d o x i n and i t s p e r f o r m i c a c i d o x i d i z e d d e r i v a t i v e w e r e u s e d a s a n t i g e n s t o t e s t w h a t e f f e c t t h e m o d i f i c a t i o n o f c y s t e i n e r e s i d u e s had on t h e p r o d u c t i o n o f a n t i b o d y . I n d i v i d u a l r a b b i t s w e r e i m m u n i z e d w i t h a d -j u v a n t p r e p a r a t i o n s o f e a c h a n t i g e n . A c o m m e r c i a l p r e p a r a t i o n o f f e r r e d o x i n was u s e d f o r t h e p r o d u c t i o n o f a n t i s e r a and o x i d i z e d f e r r e d o x i n was p r e p a r e d f r o m t h i s a s d e s c r i b e d i n M e t h o d s . Serum was c o l l e c t e d and t h e p r e s e n c e o f s p e c i f i c a n t i b o d i e s was t e s t e d f o r by means o f t h e c o m p l e m e n t f i x a t i o n r e a c t i o n . T h i s m e t h o d was c h o s e n f o r i n i t i a l d e t e r m i n a t i o n o f a n t i b o d y s i n c e i t i s t h e m o s t s e n s i t i v e i n v i t r o m e t h o d o f d e t e c t i o n a v a i l a b l e , and w i l l i n d i c a t e an a n t j g e n -a n t i b o d y r e a c t i o n when o t h e r m e t h o d s , s u c h a s h e m a g g l u t i n a t i o n o r p r e c i p i t a t i o n a r e n e g a t i v e ( K a b a t a n d M a y e r , 1962). E a c h a n t i s e r u m was a s s a y e d w i t h o n e c o n c e n t r a t i o n o f i t s homo-l o g o u s a n t i g e n o v e r a r a n g e o f 10 c o m p l e m e n t d i l u t i o n s , w h i c h w e r e p r e v i o u s l y f o u n d t o g i v e a good r a n g e o f h e m o l y s i s w i t h t h e i n d i c a t i n g s y s t e m . E n d p o i n t s w e r e c a l c u l a t e d a s t h e 50% h e m o l y s i s l e v e l , s i n c e t h e r e a c t i o n has a l i n e a r r e l a t i o n s h i p w i t h c o m p l e m e n t c o n c e n t r a t i o n i n t h i s r a n g e , w h i l e t o w a r d 100% h e m o l y s i s t h e r e l a t i o n s h i p b e g i n s t o l e v e l o f f . B o t h n a t i v e Fd and OFd w e r e f o u n d t o be a n t i g e n i c i n r a b b i t s . O x i d i z e d f e r r e d o x i n p r o v e d t o be a s t r o n g e r a n t i g e n , p r o -d u c i n g a t i t e r o f 1/200, w h i l e n a t i v e Fd a n t i s e r u m had a t i t e r o f 1A0. T h e p r e c i p i t a t i n g a b i l i t i e s o f t h e two a n t i s e r a w e r e a l s o t e s t e d . In t h i s c a s e , a n t i s e r u m a t a 1 / 4 . d i l u t i o n was m i x e d w i t h v a r y i n g c o n c e n t r a t i o n s o f h o m o l o g o u s a n t i g e n and t h e r e s u l t i n g p r e c i p i t a t e s w e r e r e m o v e d , w a s h e d and d i s s o l v e d i n d i l u t e NaOH. . Q u a n t i t a t i o n i n v o l v e d d e t e r m i n i n g t h e a b s o r b a n c e o f t h e s o l u t i o n a t 280 nru. N a t i v e Fd a n t i s e r u m e v e n when u n d i l u t e d , f a i l e d t o p r e c i p i t a t e i t s h o m o l o g o u s a n t i g e n , p o s s i b l y due t o i t s low t i t e r . OFd a n t i s e r u m d i d p r e c i p i t a t e "its h o m o l o g o u s a n t i g e n , a n d t h e r e s u l t s a r e p r e s e n t e d g r a p h i c a l l y i n F i g u r e 5-I I I . M o d i f i c a t i o n o f t h e C y s t e i n e R e s i d u e s o f F e r r e d o x i n A f t e r e s t a b l i s h i n g t h e a n t i g e n i c i t y o f f e r r e d o x i n , t h e a r e a s o f 42 Fig. 5 . Quantitative precipitation reactions of antiserum against OFd at 1:4 with various antigenic preparations: 0-© native f e r r e d o x i n ; - ^ TCA-Fd; 0-0 OFd. t h e m o l e c u l e r e s p o n s i b l e f o r b i n d i n g s p e c i f i c a n t i b o d y w e r e i n v e s t i -g a t e d . The g e n e r a l m e t h o d u s e d f o r d e t e r m i n i n g a n t i g e n i c d e t e r m i n a n t s , i n v o l v i n g i s o l a t i o n o f h a p t e n i c p e p t i d e s f r o m a p r o t e o l y t i c d i g e s t , was n o t f o u n d t o be p r a c t i c a b l e f o r t h e f e r r e d o x i n m o l e c u l e . A l -t h o u g h t h e p r o t e i n c o n t a i n s one r e s i d u e w h i c h s h o u l d be 3 s u s c e p t i b l e t o t r y p s i n ( L y s ) a n d two w h i c h s h o u l d be s u s c e p t i b l e t o 2 30 c h y m o t r y p s i n ( T y r and Phe ) , ( F i g u r e 6), t h e n a t i v e m o l e c u l e i s c o m p l e t e l y r e s i s t a n t t o h y d r o l y s i s by t h e s e e n z y m e s ( T a n a k a , 1 9 6 3 ) . The p e r f o r m i c a c i d o x i d i z e d p r o t e i n c a n be d i g e s t e d w i t h c h y m o t r y p s i n , i f d i g e s t i o n i s p r o l o n g e d and a l a r g e amount o f enzyme i s u s e d (35% by w e i g h t ) . In t h i s c a s e t h r e e p e p t i d e s a r e p r o d u c e d , an N H ^ - t e r m i n a l d i p e p t i d e , and two o t h e r p e p t i d e s o f a l m o s t e q u a l s i z e (28 a n d 25 r e s i d u e s ) and c h a r g e . The f i r s t p e p t i d e i s t o o s m a l l t o be i m m u n o l o g i c a l l y u s e f u l a n d t h e l a t t e r two a r e t o o s i m i l a r i n s i z e and c h a r g e t o make s e p a r a t i o n f e a s i b l e . T h e c y s t e i n e r e s i d u e s i n t h e l i g a n d - f r e e a p o p r o t e i n c a n be m o d i f i e d t o make them s u s c e p t i b l e t o t r y p s i n o r c h y m o t r y p s i n , and t h i s m e t h o d i n f a c t , was u s e d t o i s o l a t e p e p t i d e s f o r s e q u e n c e a n a l y s i s ( T a n a k a e t a 1 ,.1966) , b u t was n o t c o n s i d e r e d u s e f u l u n t i l t h e r o l e o f c y s t e i n e i n a n t i - • g e n i c i t y was d e t e r m i n e d . T h e r e f o r e , t h e s e c o n d common m e t h o d u s e d f o r d e t e r m i n i n g a n t i g e n i c b i n d i n g s i t e s was c h o s e n f o r i n i t i a l s t u d i e s . . T h i s i n v o l v e s m o d i f i -c a t i o n o f s p e c i f i c r e s i d u e s i n t h e p r o t e i n and s u b s e q u e n t t e s t i n g o f t h e m o d i f i e d a n t i g e n f o r i t s a b i l i t y t o b i n d a n t i b o d y . F e r r e d o x i n i s u n i q u e i n t h a t i t c o n t a i n s 8 c y s t e i n e r e s i d u e s , a h i g h p r o p o r t i o n 1 10 Ala-Tyr-Lys-Ile-Ala-Asp-Ser-Cys-Val-Ser-Cys-Gly-Ala-Cys-Ala-Ser-Glu-Cys 20.' 30 -Pro-Val-Asn-Ala-Ile-Ser-Gln-Gly-Asp-Ser-Ile-Phe-Val-Ile-Asp-Ala-Asp-Thr kO 50 •Cys-Ile-Asp-Cys-Gly-Asn-Cys-Als-Asn-Val-Cys-PRO-VAL-GLY-ALA-PRO-VAL-GLN-GLU Figure 6:. Amino Acid Sequence of C. pasteurianum Ferredoxin. Suggested antigenic sequences are underlined. Upper Case: C-terminal octapeptide. f o r s u c h a s m a l l m o l e c u l e . T h e s e a r e n o t i n v o l v e d i n d i s u l f i d e b o n d s and a r e s p a c e d r e l a t i v e l y e v e n l y i n two r e g i o n s o f t h e m o l e c u l e ( F i g u r e 6). T h r e e d e r i v a t i v e s o f n a t i v e f e r r e d o x i n w e r e p r e p a r e d i n o r d e r t o d e t e r m i n e t h e p a r t i c i p a t i o n o f c y s t e i n e r e s i d u e s i n t h e a n t i g e n i c i t y o f t h e p r o t e i n . In t h e n a t i v e m o l e c u l e , t h e 8 m o l e c u l e s o f i r o n and h y d r o g e n s u l f i d e c o m p l e x e d t o e a c h p r o t e i n ( B u c h a n a n et_ a j _ , 1963) a r e p o s t u l a t e d t o be c o m p l e x e d t o t h e p r o t e i n t h r o u g h t h e s u l f h y d r y l g r o u p s o f t h e c y s t e i n e r e s i d u e s ( E h r e n b e r g e t a 1 , 1967) s o t h a t r e m o v a l o f t h e i r o n and h y d r o g e n s u l f i d e w o u l d be e x p e c t e d t o d i s r u p t t h e e n v i r o n m e n t o f t h e c y s t e i n e r e s i d u e s o f t h e n a t i v e m o l e c u l e . T r e a t m e n t o f n a t i v e f e r r e d o x i n w i t h 5% t r i c h l o r o -a c e t i c a c i d c a u s e d i m m e d i a t e b l e a c h i n g a n d p r e c i p i t a t i o n o f t h e p r o t e i n a c c o m p a n i e d by e v o l u t i o n o f H^S, phenomena a s s o c i a t e d w i t h r e m o v a l o f t h e i n o r g a n i c l i g a n d s f r o m t h e p r o t e i n ( T a n a k a e t _ ' a l _ , 1 9 6 3 ) . From 30 mg o f n a t i v e f e r r e d o x i n , 23 mg o f w h i t e a p o f e r r e d o x i h ( J C A - F d ) w e r e o b t a i n e d w h i c h w e r e r e a d i l y s o l u b l e i n d i s t i l l e d w a t e r o r b u f f e r . The o n l y p r e c a u t i o n t a k e n t o e n s u r e t h a t d i s u l f i d e b o n d ' f o r m a t i o n d i d n o t o c c u r i n t h e T C A - p r e c i p i t a t e d p r o d u c t , was t h e a d d i t i o n o f 0 . 1 0 ml o f 2 - m e r c a p t o e t h a n o l t o t h e o r i g i n a l p r e c i p i t a t i o n m i x t u r e . T h i s p r e c a u t i o n seemed a d e q u a t e , s i n c e i n s o l u b l e a g g r e g a t e s w e r e n e v e r o b s e r v e d i n t h e p r e p a r a t i o n , and i t s i m m u n o l o g i c a l r e a c t i v i t y r e m a i n e d u n i f o r m . f r o m t e s t t o t e s t . Any a l t e r a t i o n s o c c u r r i n g due t o d i -s u l f i d e f o r m a t i o n w o u l d be e x p e c t e d t o d e c r e a s e i t s i m m u n o l o g i c a l r e a c t i v i t y a n d , a s w i l l be shown b e l o w , t h i s d i d n o t o c c u r . A f t e r h y d r o l y s i s w i t h 6 N HC1, t h e a m i n o a c i d c o m p o s i t i o n o f a s a m p l e o f T C A - F d was d e t e r m i n e d on a ' B e c k m a n A m i n o A c i d A n a l y z e r . The c o m p o s i t i o n i s l i s t e d i n T a b l e 2. A f t e r h y d r o l y s i s , a m i n o a c i d a n a l y s i s showed t h a t t h e c y s t e i n e r e s i d u e s w e r e t o t a l l y c o n v e r t e d t o c y s t e i c a c i d . S i n c e , a s w i l l be r e p o r t e d b e l o w , t h i s s a m p l e o f T C A - F d r e a c t e d w i t h i o d o a c e t a m i d e , i n d i c a t i n g t h e p r e s e n c e o f f r e e s u l f h y d r y l g r o u p s , and s i n c e i t s i m m u n o l o g i c a l b e h a v i o u r d i f f e r e d q u a n t i t a t i v e l y f r o m an a u t h e n t i c s a m p l e o f o x i d i z e d f e r r e d o x i n , I t was a s s u m e d t h a t t h e o x i d a t i o n o f t h e s u l f h y d r y l g r o u p s was t h e r e s u l t o f h y d r o l y s i s , p o s s i b l y d u e t o t h e p r e s e n c e o f t r a c e a m o u n t s o f t r i c h l o r o a c e t i c a c i d . Two f u r t h e r d e r i v a t i v e s w e r e p r e p a r e d f r o m t h e T C A - t r e a t e d p r o d u c t ; o n e in w h i c h t h e c y s t e i n e r e s i d u e s w e r e c o n v e r t e d t o c y s t e i c a c i d by p e r f o r m i c a c i d o x i d a t i o n t o p l a c e a s t r o n g n e g a t i v e c h a r g e on t h e r e s i d u e s ; and o n e i n w h i c h t h e s u l f h y d r y l g r o u p s w e r e a l k y -l a t e d w i t h i o d o a c e t a m i d e t o p l a c e a b u l k y n e u t r a l g r o u p on t h e r e s i d u e s . B o t h p r e p a r a t i o n s w e r e r e a d i l y s o l u b l e i n d i s t i l l e d w a t e r and b u f f e r . T h e d e g r e e o f m o d i f i c a t i o n o f t h e c y s t e i n e r e s i d u e s was d e t e r m i n e d by a m i n o a c i d a n a l y s i s a n d c a l c u l a t i o n o f t h e a m o u n t s o f c y s t e i c a c i d a n d c a f b o x a m i d o m e t h y l c y s t e i n e , r e s p e c t i v e l y , w h i c h w e r e r e -c o v e r e d . T h e a m i n o a c i d c o m p o s i t i o n s o f t h e two d e r i v a t i v e s a r e l i s t e d i n T a b l e 2, and show t h a t t h e r e a c t i o n i n e a c h c a s e was v i r t u a l l y c o m p l e t e , s i n c e no f r e e c y s t e i n e c o u l d be r e c o v e r e d . ( V . I m m u n o l o g i c a l R e a c t i v i t y o f F e r r e d o x i n and . i t s D e r i v a t i v e s The e x t e n t o f c r o s s - r e a c t i o n b e t w e e n t h e two a n t i s e r a and t h e f o u r d i f f e r e n t f e r r e d o x i n p r e p a r a t i o n s ( n a t i v e F d , T C A - F d , OFd and CAM-Fd) was d e t e r m i n e d by c o m p l e m e n t f i x a t i o n . N a t i v e f e r r e d o x i n a n t i s e r u m a t a d i l u t i o n o f 1/40 was m i x e d w i t h v a r y i n g a m o u n t s o f e a c h p r e p a r a t i o n o v e r a r a n g e o f 10 c o m p l e m e n t d i l u t i o n s . E a c h t e s t was p e r f o r m e d a t l e a s t t w i c e and t h e y w e r e f o u n d t o a g r e e w i t h i n 5%. The e n d p o i n t s w e r e c a l c u l a t e d a s t h e 50% h e m o l y s i s p o i n t f r o m p r o b i t p l o t s o f a c t u a l p e r c e n t a g e s . As shown i n F i g u r e 6 , c r o s s -r e a c t i o n o c c u r r e d i n a l l c a s e s . T h e r e s u l t s a r e s u r p r i s i n g h o w e v e r , i n t h a t t h e m a x i m a l r e a c t i o n s w e r e o b t a i n e d w i t h t h e i r o n s u l f i d e -f r e e a n d a l k y l a t e d d e r i v a t i v e s , r a t h e r t h a n w i t h t h e s u p p o s e d l y h o m o l o g o u s a n t i g e n , n a t i v e f e r r e d o x i n . T h e f a c t t h a t t h e two n e u t r a l , 1 i g a n d - f r e e p r e p a r a t i o n s a p p e a r t o r e a c t m a x i m a l l y w i t h t h e a n t i b o d y may i n d i c a t e t h a t i n v i v o t h e n a t i v e m o l e c u l e i s m o d i f i e d b e f o r e a n t i b o d i e s a r e p r o d u c e d a g a i n s t i t . OFd a n t i s e r u m w a s . a s s a y e d a t a . 1 / 2 0 0 d i l u t i o n iin a s i m i l a r manner t o t h e n a t i v e f e r r e d o x i n a n t i s e r u m , and a g a i n , a s shown i n F i g u r e 7 , c r o s s - r e a c t i o n o c c u r r e d i n a l l c a s e s , w i t h t h e h o m o l o g o u s s y s t e m g i v i n g t h e m a x i m a l r e a c t i o n . T h e good c r o s s - r e a c t i o n o b s e r v e d w i t h OFd a n t i s e r u m i s i n t e r e s t i n g i n l i g h t o f t h e r e s u l t s o f Brown ( 1 9 6 2 ) s t u d y i n g t h e a n t i g e n i c i t y o f p e r f o r m i c a c i d o x i d i z e d r i b o n u c l e a s e . T h e c y s t e i c a c i d r e s i d u e s w o u l d be e x p e c t e d t o be h i g h l y immuno-05 1.0 ug ANTIGEN 5.0 10.0 F i g . J7. Complement f i x a t i o n r e a c t i o n o f a n t i s e r u m a g a i n s t n a t i v e f e r r e d o x i n at 1:1+0 w i t h v a r i o u s a n t i g e n i c p r e p a r a t i o n s ; 9 - 9 n a t i v e F d ; t\ - £\ T C A - F d , A - A a l k y l a t e d F d ; 0 - 0 O F d . 1= 10 • 0.5 1.0 5.0 100 jjg ANTIGEN F i g . 8. Complement f ixa t ion reactions of antiserum against O-Fd at 1:200 with various antigenic preparations: © - © native Fd, A - A TCA-Fd, A - A a lkylated Fd , 0 - 0 OFd. g e n i e s i n c e t h e y w o u l d be n o v e l t o t h e a n t t b o d y s y n t h e s i z i n g c e l l s , b u t he f o u n d t h a t c y s t e t c a c i d was n o t h i g h l y i m m u n o g e n i c , w i t h o n l y one o f e i g h t c y s t e i c a c i d r e s i d u e s and none o f t h e two m e t h i o n i n e s u l f o i i e r e s i d u e s a p p e a r i n g i n t h e s e q u e n c e s o f t h e two h a p t e n i c p e p t i d e s he i s o l a t e d . W i t h t h i s a n t i s e r u m , t h e c r o s s - r e a c t i o n d a t a w e r e c o n f i r m e d by t h e q u a n t i t a t i v e p r e c i p i t a t i o n r e a c t i o n u s i n g n a t i v e Fd and T C A - F d a s h e t e r o l o g o u s a n t i g e n s , (due t o l a c k o f m a t e r i a l CAM-Fd was n o t t e s t e d ) . A g a i n , c r o s s - r e a c t i o n was o b t a i n e d i h a l l c a s e s , w i t h t h e h o m o l o g o u s s y s t e m d e m o n s t r a t i n g t h e m a x i m a l r e a c t i o n , a s shown i n F i g u r e 5, w h i l e n a t i v e Fd and T C A - F d g a v e l e s s e r b u t r o u g h l y e q u a l r e a c t i o n s , p r e c i p i t a t i n g 50% a s much a n t i b o d y as O F d . T h e c r o s s - r e a c t i o n d a t a f r o m b o t h c o m p l e m e n t f i x a t i o n and p r e -c i p i t a t i o n t e s t s i n d i c a t e t h a t a t l e a s t two a n t i g e n i c s i t e s e x i s t on t h e f e r r e d o x i n m o l e c u l e w h i c h do n o t d i r e c t l y i n v o l v e c y s t e i n e , s i n c e two b i n d i n g s i t e s on t h e a n t i g e n a r e n e c e s s a r y f o r l a t t i c e f o r m a -t i o n and p r e c i p i t a t i o n . The p o s s i b l e i n v o l v e m e n t o f c y s t e i n e i n a n t i g e n i c s i t e s c a n n o t be c o m p l e t e l y e x c l u d e d , h o w e v e r , s i n c e d i f -f e r e n c e s i n r e a c t i v i t y do e x i s t b e t w e e n t h e f o u r . p r e p a r a t i o n s a n d t h e two a n t i s e r a ; a l t h o u g h t h e s e d i f f e r e n c e s may be due a t l e a s t i n p a r t , a t o u n a v a o i d a b l e c o n f i g u r a t i o n a 1 c h a n g e s c o h e j m i t t a n t w i t h c h e m i c a l m o d i f i c a t i o n . T h e c r o s s - r e a c t i o n t h a t Is o b s e r v e d i s a s s u m e d t o be t h e r e s u l t o f . r e a c t i o n o f t h e r e s p e c t i v e d e r i v a t i v e s and n o t due t o p a r t i c i p a t i o n o f u n m o d i f i e d m a t e r i a l , s i n c e a m i n o a c i d a n a l y s e s o f e a c h p r e p a r a t i o n i n d i c a t e d v i r t u a l l y c o m p l e t e c o n v e r s i o n t o t h e r e s p e c t i v e m o d i f i e d d e r i v a t i v e . V . S o l i d P h a s e S y n t h e s i s o f t h e C O O H - T e r m i n a l O c t a p e p t i d e The r e s u l t s f r o m c r o s s - r e a c t i o n b e t w e e n OFd and n a t i v e Fd a n t i -s e r a w i t h t h e f o u r p r e p a r a t i o n s o f f e r r e d o x i n i n d i c a t e t h a t a t l e a s t two a n t i g e n i c s i t e s a r e p r e s e n t on t h e p r o t e i n w h i c h do n o t h a v e c y s t e i n e r e s i d u e s i n t h e i r s e q u e n c e s . A s s u m i n g a minimum a n t i g e n i c s i z e o f 5 a m i n o a c i d r e s i d u e s , t h e a r e a s o f t h e p r o t e i n w h i c h c o u l d , a c t a s a n t i b o d y b i n d i n g s i t e s a r e somewhat 1 i m i t e d . The a r e a s e n -7 19 37 48 c o m p a s s i n g r e s i d u e s C y s t o C y s and C y s t o C y s c a n be e l i m i n a t e d , l e a v i n g t h r e e r e g i o n s , f r o m A l a ^ t o S e r ^ , P r o ' ^ t o T h r 3 6 and P r o ^ 55 t o G l u , w h i c h p o s s i b l y p o s s e s s a n t i g e n i c a c t i v i t y ( F i g u r e A t t h i s p o i n t , i t was c o n s i d e r e d a d v a n t a g e o u s t o o b t a i n p e p t i d e s by s o l i d p h a s e s y n t h e s i s r a t h e r t h a n by enzyme d i g e s t i o n o f a c y s t e t n e -m o d i f i e d d e r i v a t i v e o f f e r r e d o x i n , s i n c e t h e s y n t h e t i c m e t h o d p r o v i d e s a p u r e r p r o d u c t i n l e s s t i m e w i t h g r e a t e r y i e l d . The f i r s t a r e a c h o s e n f o r s t u d y was t h e C O O H - t e r m i n a l o c t a p e p t i d e o f s e q u e n c e p r o - v a l -g l y - a l a - p r o - v a l - g m r g l u b e c a u s e i t i s r e l a t i v e l y s m a l l t h o u g h l a r g e v.. __ .^*r e n o u g h t o p o s s e s s a n t i g e n i c a c t i v i t y , i t i s q u i t e h y d r o p h o b i c , i t i s e a s i l y s y n t h e s i z e d ( c o n t a i n i n g o n l y o n e r e s i d u e w i t h a r e a c t i v e s i d e g r o u p ) , and f i n a l l y , t h e C O O H - t e r m i n a l p o r t i o n s o f s p e r m w h a l e m y o -g l o b i n and hen e g g - w h i t e l y s o z y m e h a v e b e e n i m p l i c a t e d i n a n t i b o d y b i n d i n g . So l t d p h a s e s y n t h e s i s o f p e p t i d e s i s a r e c e n t t e c h n i q u e d e s c r i b e d by M e r r i f i e l d (1964). I t i n v o l v e s t h e s t e p w i s e a d d i t i o n o f a m i n o a c i d s t o a g r o w i n g p e p t i d e c h a i n w h i c h i s a t t a c h e d a t i t s C O O H - t e r m i n a l end t o an i n s o l u b l e s u p p o r t , i n t h i s c a s e a r e s i n bead o f c h l o r o -m e t h y l a t e d p o l y s t y r e n e - d i v i n y l b e n z e n e c o p o l y m e r : T h e f i r s t a m i n o a c i d , as i t s t r i e t h y l a m m o n i u m s a l t , i s a t t a c h e d t o t h e r e s i n by an e s t e r l i n k v i a t h e c h l o r o m e t h y l g r o u p s . T h e N H ^ - t e r m i n a l e n d o f e a c h a m i n o a c i d i s b l o c k e d by a t e r t - b u t y l o x y c a r b o n y l g r o u p ( t - b o c ) t o e l i m i n a t e s i d e r e a c t i o n s . A t t a c h m e n t o f t h e g r o w i n g p e p t i d e t o an i n s o l u b l e s u p p o r t f a c i l i t a t e s r e m o v a l o f s o l v e n t s and e x c e s s r e a g e n t s by s i m p l e f i l t r a t i o n , e l i m i n a t i n g w a s t e f u l a n d t i m e - c o n s u m i n g p u r i f i -c a t i o n p r o c e d u r e s a t e a c h s t e p . T h e t - b o c g r o u p o f t h e t e r m i n a l a m i n o a c i d i s r e m o v e d by 1 N HCl i s g l a c i a l a c e t i c a c i d and t h e n e x t a m i n o a c i d ' i s c o u p l e d t o t h e f r e e d N H 2 ~ t e r m i n a l g r o u p u s i n g d i -c y c l o h e x y l c a r b o d i i m i d e (DCC) a s a d e h y d r a t i n g a g e n t i n f o r m i n g t h e p e p t i d e b o n d . T h e a m i n o a c i d s a s p a r a g i n e and g l u t a m i n e a r e c o u p l e d a s t h e i r p - n i t r o p h e r i y l e s t e r s w i t h o u t t h e m e d i a t i o n o f D C C , i n w h i c h c a s e t h e r e a c t i o n t i m e was i n c r e a s e d f r o m 2 t o 5 h o u r s and a 5 - f o l d i n s t e a d o f a 3 . 5 - f o l d m o l a r e x c e s s o f t h e a m i n o a c i d was a d d e d . T h e p e p t i d e i s b u i l t up s t e p w i s e i n t h i s m a n n e r , w i t h r e a c t i v e R' g r o u p s s u i t a b l y b l o c k e d , g e n e r a 1 l y by a b e n z y l g r o u p . A f t e r t h e l a s t a m i n o a c i d i s a t t a c h e d , t h e p e p t i d e i s c l e a v e d f r o m t h e r e s i n by a n h y d r o u s HBr i n a n h y d r o u s t r i f l u o r a c e t i c a c i d . T h i s t r e a t m e n t a l s o r e m o v e s t h e f i n a l t - b o c g r o u p and t h e 0 - b e n z y l p r o t e c t i v e Fig. 9- Flow chart of Solid Phase Peptide Synthesis. CH3 O H R, O I II - I I II CH 3 —C—O—C—N—C—C—O - + CI—CH,—£ ^ — POLYMER CH3 H Boc amino acid chloromethyl polymer CH3 O H R i O POLYMER CH3 CH. il CH3—C I CH3. isobutylene H Boc amino acyl polymer HC1—dioxane; Et3N DEPROTECT: NEUTRALIZE H Ri O I I II + CO, + H — N — C — C — O — C H 2 —n amino acyl polymer Boc amino acid I diimide POLYMER COUPLE CH3 O H R 2 O H R i O I II I I II I I II CH 3—C—O—C—N—C—C—N—C—C—O—CH POLYMER CH3 H H Boc peptide polymer 7 HBr—F3CCOOH CLEAVE. CH, H R. O H R, O • I I I I. II I I II /=\ r CH3—C + CO, + HN—C—C—N—C—C—OH -f Br—CH,—<^^—[ POLYMER CH 3 isobutylene H H peptide 'Reproduced from Solid Phase Peptide Synthesis by John Stewart and Janis Young, p. 3. g r o u p p r e s e n t on g l u t a m i c a c i d o f t h e p e p t i d e s e q u e n c e d i n t h i s i n s t a n c e . T h e s e q u e n c e o f s t e p s i s r e p r e s e n t e d d i a g r a m m a t i c a l l y i n F i g u r e 10. T h e amount o f t h e f i r s t a m i n o a c i d a t t a c h e d t o t h e r e s i n i s d e t e r m i n e d . b y h y d r o l y s f s o f a w e i g h e d s a m p l e f o l l o w e d by a m i n o a c i d a n a l y s i s , and f r o m t h i s t h e amount o f e a c h s u b s e q u e n t a m i n o a c i d and DCC t o be a d d e d i s c a l c u l a t e d . T h e c l e a v e d p e p t i d e was p u r i f i e d by i o n - e x c h a n g e c h r o m a t o g r a p h y and t h e p e a k c o n t a i n i n g t h e p e p t i d e w i t h t h e b e s t a m i n o a c i d r a t i o was i d e n t i f i e d by a m i n o a c i d a n a l y s i s . T h e e l u t i o n p r o f i l e i s shown i n F i g u r e 11, a n d t h e a m i n o a c i d c o m p o s i t i o n o f t h e p u r i f i e d p e p t i d e i s l i s t e d i n T a b l e 3- T h e c o m p o s i t i o n i s v e r y c l o s e t o t h e t h e o r e t i -c a l . T h i s , and t h e f a c t t h a t o n l y o n e n i h h y d r i n - p o s i t i v e s p o t c o u l d be d e t e c t e d by h i g h v o l t a g e e l e c t r o p h o r e s i s i n d i c a t e d t h a t t h e p e p t i d e was e s s e n t i a l l y h o m o g e n e o u s . Of a p o s s i b l e 0.72 m i c r o m o l e s , 0.267 m i c r o m o l e s o f g l u t a m i c a c i d was a t t a c h e d t o t h e r e s i n (36.8% o f t h e t h e o r e t i c a l ) , and 100 m i c r o m o l e s o f p e p t i d e was r e c o v e r e d , w h i c h i s 37-5% o f t h e t h e o r e t i c a l y i e l d c a l c u l a t e d f r o m t h e amount o f a t t a c h m e n t . V I . I m m u n o l o g i c a l T e s t i n g o f t h e S y n t h e t i c P e p t i d e The h a p t e n i c a c t i v i t y o f t h e s y n t h e t i c p e p t i d e was d e t e r m i n e d by t e s t i n g i t s a b i l i t y t o i n h i b i t c o m p l e m e n t f i x a t i o n . V a r y i n g a m o u n t s o f t h e p e p t i d e w e r e a d d e d t o a s e r i e s o f t u b e s c o n t a i n i n g n a t i v e Fd a n t i s e r u m and OFd a n t i s e r u m a t ]/k0 a n d 1/200 d i l u t i o n s r e s p e c t i v e l y . ML ELUENT F i g . 10. E l u t i o n p r o f i l e o f t h e COOH-terminal p e p t i d e . The a r r o w i n d i c a t e s t i m e o f t h e b u f f e r change a f t e r c o m p l e t i o n o f t h e g r a d i e n t . The peak e l u t i n g a t about 105 ml. c o n t a i n e d t h e o c t a p e p t i d e . 56 Amino Acid umoles molar ratio residues Glutamic acid 1.121 2.19 . 2 Proline 1.033 2.02 2 Glycine 0.476 0.93 1 Alanine 0.5V7 1.07 1 Valine 1.025 2.00 2 Table 3. Amino acid analysis of the C-terminal octapeptide of ferredoxin, sequence: pro-val-gly-ala-pro-val-gln-glu. A f t e r i n c u b a t i o n , h o m o l o g o u s a n t i g e n and c o m p l e m e n t w e r e a d d e d , and t h e n t h e d e g r e e o f f i x a t i o n was d e t e r m i n e d f r o m t h e amount o f h e m o l y s i s . T h e d e g r e e o f i n h i b i t i o n was c a l c u l a t e d a s t h e d i f f e r e n c e i n p e r c e n t a g e h e m o l y s i s b e t w e e n t h e t e s t s e r i e s a n d t h e t u b e c o n -t a i n i n g t h e same c o n c e n t r a t i o n o f c o m p l e m e n t i n t h e p o s i t i v e c o n t r o l ( n o t c o n t a i n i n g h a p t e n ) when c o m p a r e d t o a no h a p t e n - n o a n t i g e n c o n t r o l . T h e a v e r a g e d r e s u l t s o f t h e s e t e s t s a r e shown g r a p h i c a l l y i n F i g u r e 1 1 . T h e o c t a p e p t i d e e x h i b i t e d i n h i b i t o r y a c t i v i t y w i t h b o t h s e r a , t o t h e e x t e n t o f 25% w i t h n a t i v e Fd a n t i s e r u m and 36% w i t h OFd a n t i s e r u m . In a l l c a s e s , t h e h a p t e n p r o v e d more i n h i b i t o r y a t 25 ug t h a n a t 50 ug; t h i s phenomenon was c o n s i s t e n t f r o m o n e s e t o f c o m p l e m e n t f i x a t i o n t e s t s t o t h e n e x t , a n d was s p e c i f i c f o r t h e f e r r e d o x i n s y s t e m , s i n c e no i n c r e a s e d f i x a t i o n c o u l d be d e t e c t e d iri a n o n - h o m o l o g o u s s y s t e m c o n s i s t i n g o f l y s o z y m e and a n t i - l y s o z y m e s e r u m when 50 ug o f h a p t e n was u s e d . The a b i l i t y o f t h e h a p t e n t o i n h i b i t t h e p r e c i p i t a t i o n r e a c t i o n b e t w e e n OFd and i t s h o m o l o g o u s a n t i b o d y was a l s o t e s t e d . tn i n i t i a l s t u d i e s , h a p t e n i n c o n c e n t r a t i o n s r a n g i n g f r o m 2 . 5 t o 50 u g In 0 . 1 ml, w e r e a d d e d t o t r i p l i c a t e s a m p l e s o f a n t i s e r u m and i n c u b a t e d f o r 1 h r , t h e n h o m o l o g o u s a n t i g e n was a d d e d and t h e i n c u b a t i o n c o n t i n u e d o v e r -n i g h t . By t h i s m e t h o d , a maximum i n h i b i t i o n o f o n l y 7% was o b t a i n e d . T h e r e f o r e , t h e t e s t was m o d i f i e d : h a p t e n was i n c u b a t e d w i t h a n t i s e r u m f o r 1 h r , w i t h a r a n g e f r o m 500 t o 2 . 5 u g , and t h e n a n t i g e n was a d d e d and i n c u b a t i o n was c o n t i n u e d . A f t e r 1 h r , t h e p r e c i p i t a t e s 58 2.5 5.0 10 25 5 0 ijg HAPTEN F i g . 11.• I n h i b i t i o n of complement f i x a t i o n by the COOH-terminal octa-peptide when tested against native ferredoxin and OFd and t h e i r homologous antisera. 0-0 and 0 - 8 represent d i l u t i o n s of 1:100 and 1:125 of complement with OFd and anti-OFd. A - 4 and A ~ represent the same d i l u t i o n s of complement with native ferredoxin and i t s homologous antigen. w e r e c e n t r i f u g e d , w a s h e d , d i s s o l v e d i n d i l u t e NaOH and t h e a b s o r b a n c e s o f t h e s o l u t i o n s r e a d a t 280 my. T h e r e s u l t s o f t h e a v e r a g e d t r i p l i -c a t e s a m p l e s a r e shown i n F i g u r e 13- The d e g r e e o f i n h i b i t i o n was s t i l l q u i t e low and t h e maximum l e v e l was n o t r e a c h e d , b u t t h e r e s u l t s w e r e r e p r o d u c i b l e t o w i t h i n 0 . 5 % F i n a l l y , t h e r e a c t i v i t y o f t h e p e p t i d e was t e s t e d by d i r e c t b i n d i n g s t u d i e s e m p l o y i n g t h e e q u i l i b r i u m d i a l y s i s t e c h n i q u e . W i t h t h i s m e t h o d , a n t i s e r u m i s s e p a r a t e d f r o m t h e h a p t e n by a s e m i -p e r m e a b l e membrane w h i c h a l l o w s p a s s a g e o f t h e s m a l l h a p t e n b u t n o t o f t h e a n t i b o d y . T h e a n t i b o d y s o l u t i o n , i n a d i a l y s i s s a c , i s i m m e r s e d i n a s o l u t i o n o f h a p t e n , and t h e h a p t e n i s a l l o w e d t o r e a c h e q u i l i b r i u m o n e a c h s i d e o f t h e membrane. A f t e r e q u i 1 i b r t u r n i s r e a c h e d t h e c o n c e n t r a t i o n o f h a p t e n on e a c h s i d e o f t h e membrane i s m e a s u r e d . T h e d i f f e r e n c e b e t w e e n t h e amount o f h a p t e n f o u n d on t h e a n t i b o d y s i d e o f t h e membrane and t h e c o n c e n t r a t i o n on t h e o u t s i d e o f t h e d i a l y s i s s a c i s a m e a s u r e o f t h e amount o f h a p t e n bound t o a n t i b o d y . In o r d e r t o m e a s u r e h a p t e n c o n c e n t r a t i o n , a r a d i o a c t i v e l a b e l , 14 1- C - A c e t y l - , was a t t a c h e d t o t h e N H 2 _ t e r m i n u s o f t h e o c t a p e p t i d e . T h i s was a c c o m p l i s h e d by l a y e r i n g a 2 0 - f o l d m o l a r e x c e s s o f l a b e l l e d a c e t i c a n h y d r i d e i n a n h y d r o u s b e n z e n e o v e r an a q u e o u s s o l u t i o n o f t h e p e p t i d e . When t h e r e a c t i o n was c o m p l e t e d , t h e e x c e s s l a b e l was s e p a r a t e d f r o m t h e o c t a p e p t i d e by p a s s i n g t h e a q u e o u s p h a s e o f t h e r e a c t i o n m i x t u r e t h r o u g h a c o l u m n o f S e p h a d e x G - 1 5 . F i g u r e 13 shows t h e e l u t i o n p r o f i l e o b t a i n e d f r o m t h i s s e p a r a t i o n . Q u a n t i t a t i o n o f 60 jug HAPTEN F i g . 12. I n h i b i t i o n of p r e c i p i t a t i o n with OFd and i t s homologous antiserum i n the presence of varying amounts of the octapeptide. 0 61 F i g . 13. The e l u t i o n p r o f i l e of the NH 2-acetylated octapeptide. The f i r s t peak contained the peptide, and the second 14 C-acetic a c i d . p e p t i d e a f t e r e l u t i o n , w a s h i n g and d r y i n g y i e l d e d a p r e p a r a t i o n c o n -t a i n i n g 3 - 5 x 10^ c p m / m i c r o m o l e . In o r d e r t o i n c r e a s e t h e t i t e r o f t h e a n t i b o d y p r e p a r a t i o n u s e d f o r e q u i l i b r i u m d i a l y s i s m e a s u r e m e n t s and t o i n c r e a s e t h e s p e c i f i c i t y o f t h e r e a c t i o n , s p e c i f i c a n t i b o d i e s w e r e i s o l a t e d f r o m t h e a n t i s e r a d i r e c t e d a g a i n s t n a t i v e f e r r e d o x i n and O F d . The s p e c i f i c i m m u n o a d s o r p t i o n method o f R o b b i n s et^ a_]_ (1967) was u s e d , i n w h i c h t h e a n t i g e n i s c o v a l e n t l y bound t o an i n s o l u b l e s u p p o r t , i n t h i s c a s e b r o m o a c e t y l c e l l u l o s e . S p e c i f i c i m m u n o a d s o r p t i o n was u s e d b e c a u s e i t p e r m i t s t h e r e m o v a l o f a n t i b o d i e s f r o m s e r u m w h i c h w i l l n o t p r e -c i p i t a t e w i t h h o m o l o g o u s a n t i g e n , a s i n t h e c a s e o f n a t t v e Fd a n t i s e r u m . The p r e p a r a t i o n o f t h e i m m u n o a d s o r b e n t i n v o l v e s two s t e p s . F i r s t , b r o m o a c e t y l b r o m i d e i s c o v a l e n t l y b o n d e d t o t h e b r o m o a c e t y l c e l l u l o s e by an e t h e r e a l l i n k , t h e n t h e a n t i g e n i s b o n d e d t o t h e b r o m o a c e t y l c e l l u l o s e by an a m i d e l i n k t h r o u g h t h e f r e e a m i n o g r o u p s p r e s e n t on t h e p r o t e i n . One o f t h e p r o b l e m s e n c o u n t e r e d i n t h i s t e c h n i q u e i s t h e b l o c k i n g o f a n t i b o d y b i n d i n g s i t e s by t h e a t t a c h m e n t o f t h e p r o t e i n t o t h e c e l l u l o s e ; t h e m e t h o d t h u s r e q u i r e s t h a t t h e b i n d i n g p o i n t s on t h e p r o t e i n be r a n d o m l y d i s t r i b u t e d i n o r d e r t o e n s u r e a d s o r p t i o n o f a l l t y p e s o f a n t i b o d y . T h e f e r r e d o x i n m o l e c u l e p r e s e n t s a s p e c i a l c a s e ' . , s j „ n c _ e , a s i d e f r o m t h e N h ^ - t e r m i n a 1 a m i n o a c i d , o n l y 3 one o t h e r f r e e a m i n o g r o u p o c c u r s i n t h e m o l e c u l e , a t L y s . T h e r e f o r e , t h e p o i n t s o f a t t a c h m e n t a r e l i m i t e d t o o n e end o f t h e p r o t e i n , l e a v i n g v i r t u a l l y t h e e n t i r e m o l e c u l e f r e e f o r a n t i b o d y b i n d i n g ( u n l e s s , o f c o u r s e , a n y a n t i b o d y was d i r e c t e d t o t h e N h ^ - t e r m i n u s o f the p r o t e i n ) . S i n c e t h e a n t i b o d y d i r e c t e d a g a i n s t t h e C 0 0 H -t e r m i n u s was t o be m e a s u r e d , t h i s was n o t c o n s i d e r e d a s e r i o u s d r a w b a c k . N a t i v e f e r r e d o x i n a n t i s e r u m and OFd a n t i s e r u m w e r e a d s o r b e d t o an O F d - c e l l u l o s e c o n j u g a t e , s i n c e , a s c o m p l e m e n t f i x a t i o n d a t a s h o w , t h e a n t i s e r u m t o n a t i v e f e r r e d o x i n r e a c t s p r e f e r e n t i a l l y w i t h a d e n a t u r e d a n t i g e n . A f t e r e l u t i o n o f t h e a d s o r b e d a n t i b o d y , b o t h p r e p a r a t i o n s w e r e b r o u g h t t o 1 5 . 0 m l , and t h e c o m p l e m e n t f i x a t i o n t i t e r s d e t e r m i n e d . The p u r i f i e d p r e p a r a t i o n s t i t e r e d a t 1/160 and 1 / 9 0 0 f o r n a t i v e f e r r e d o x i n a n t i s e r u m and OFd a n t i s e r u m , r e s p e c t i v e l y . When c o m p a r e d t o t h e o r i g i n a l t i t e r s o f 1/40 and 1/200 t h i s r e p r e s e n t s a r e c o v e r y o f 8 3 . 3 % and 9 0 % , r e s p e c t i v e l y , w h i c h a r e w i t h i n t h e r a n g e o f 8 0 - 9 0 % r e c o v e r y r e p o r t e d by R o b b i n s et_ aj_ ( 1 9 6 7 ) . T h e c o n c e n t r a t i o n s o f p r o t e j n ' w e r e 420 p g / m l f o r OFd a n t i b o d y and 240 u g / m l f o r f e r r e d o x i n a n t i b o d y . I t i s i n t e r e s t i n g t o n o t e t h a t w h i l e t h e r e was a f i v e - f o l d ' d i f f e r e n c e i n t i t r e b e t w e e n t h e p u r i f i e d p r e p a r a t i o n s o f OFd and n a t i v e Fd a n t i b o d y , t h e r e was a l e s s t h a n t w o - f o l d d i f f e r e n c e i n t h e p r o t e i n c o n c e n t r a t i o n . T h i s may r e f l e c t e i t h e r a d e c r e a s e d a v i d i t y i n t h e a n t i b o d y p o p u l a t i o n o f Fd a n t i s e r u m , o r t h a t a l a r g e p a r t o f t h e a n t i b o d y p o p u l a t i o n o f Fd a n t i s e r u m i s d i r e c t e d t o a d e r i v a t i v e o t h e r t h a n n a t i v e f e r r e d o x i n . T h i s q u e s t i o n c a n n o t be s e t t l e d u n t i l t h e f a t e o f n a t i v e f e r r e d o x i n i n v i v o h a s been e s t a b l i s h e d , a l t h o u g h f r o m c r o s s - r e a c t i o n d a t a i t a p p e a r s t h a t antibodies are preferentially directed against a denatured, ligand-free derivative. The equilibrium dialysis method of determining reaction between hapten and antibody may be considered the best available, since it measures direct binding, whereas other methods, such as inhibition of precipitation and of complement fixation, measure a negative quantity, allowing greater interference due to non-specific effects. It is also a sound way to measure the association constant of the interaction between the hapten and the antibody binding site since the measurements are made at equilibrium. The following equation expresses the interaction between a low molecular weight hapten and its antibody and the derivation of the association constant: H + B = HB (HB) = K (H) CB) The molar concentration of antibody and bound and free hapten must be determined in order to establish K q . The amount of free hapten can be determined at equilibrium by doubling the concentration in a given volume found on the antibody-free side of the dialysis membrane, and by subtracting this quantity from the concentration of hapten iri the same volume originally added, the quantity of the bound hapten in a given volume of antibody solution, can be determined. Expressed on a molar basis, it is possible to calculate the ratio of the number of moles of hapten bound per mole antibody and the c o n c e n t r a t t o n o f f r e e h a p t e n . In t h e e x p e r i m e n t p e r f o r m e d h e r e , l a b e l l e d h a p t e n a t t w o c o n c e n t r a t i o n s , ( 0 . 0 0 1 and 0 . 0 0 2 m i c r o m o l e s / m l ) was d i a l y s e d a g a i n s t p u r i f i e d a n t i b o d i e s e l i c i t e d a g a i n s t OFd a n d n a t i v e F d , and a g a i n s t n o n - s p e c i f i c gamma g l o b u l i n and P B S , f o r h d a y s a t h C , a t w h i c h t i m e e q u i l i b r i u m was r e a c h e d . The r e s u l t s f r o m t h e e q u i l i b r i u m d i a l y s i s e x p e r i m e n t a r e g i v e n i h T a b l e k. By t h e c r i t e r i o n s t a t e d a b o v e ( e x c e s s h a p t e n a s s o c i a t e d w i t h t h e a n t i b o d y s o l u t i o n a t e q u i l i b r i u m ) , t h e o c t a p e p t i d e b i n d s w i t h b o t h OFd and n a t i v e F d ' a n t i s e r a . W h i l e t h e number o f e x c e s s c o u n t s a r e T o w , t h e y a r e c o n s i s t e n t l y h i g h e r t h a n f r e e h a p t e n , a n d g r e a t e r t h a n t h a t a s s o c i a t e d w i t h n o n - s p e c i f i c gamma g l o b u l i n . S i n c e no e f f o r t was t a k e n t o s e p a r a t e l a b e l l e d f r o m u n l a b e l l e d p e p t i d e , and s i n c e more t h a n t h r e e c o n c e n t r a t i o n s o f h a p t e n a r e r e q u i r e d , t h e K q a n d p e r c e n t a g e o f a n t i b o d y i n v o l v e d i n h a p t e n b i n d i n g w e r e n o t c a l c u l a t e d . B i n d i n g was d e m o n s t r a t e d b e t w e e n t h e o c t a p e p t i d e and b o t h a n t i s e r a . T h i s , a l o n g w i t h t h e d a t a o b t a i n e d f r o m h a p t e n i n h i b i t i o n t e s t s , i n d i c a t e s t h a t t h e C O O H - t e r m i n a 1 o c t a p e p t i d e . o f C . p a s t e u r i a n u m f e r r e d o x i n , i s an a r e a o f a n t i g e n i c i t y o n t h e p r o t e i n . I t I s i n t e r e s t i n g t o n o t e t h a t t h o u g h OFd and n a t i v e f e r r e d o x i n a n t i s e r a w e r e e v o k e d by c h e m i c a 1 l y . q u i t e d i f f e r e n t a n t i g e n s , b o t h c o n t a i n e d a n t i b o d i e s d i r e c t e d a g a i n s t t h e same p o r t i o n o f t h e p r o t e i n . T h i s o b s e r v a t i o n i s i n t e r e s t i n g i n - t h e l i g h t o f t h e r e s u l t s o b t a i n e d by G e r w i n g a n d Thompson ( 1 9 6 9 ) on r e d u c e d and c a r b o x y m e t h y 1 a t e d l y s o z y m e . T h e y COUNTS PER MIN. 0.001 mc.mole/ml. . 0.002 mc.mole/ml. Inside Outside Difference Inside Outside Difference Fd Antiserum 656 618 38 1212 1150 62 OFd Antiserum 69k 639 55 1360 1255 105 Gamma gl o b u l i n 673 66k 9 1295 1299 -5 Table k. E q u i l i b r i u m d i a l y s i s data. f o u n d t h a t a p e p t i d e p o s s e s s i n g h a p t e n i c a c t i v i t y i n v o l v e d t h e a r e a o f t h e p r o t e i n f o u n d by S h i n k a e t a_l_ (1967) t o be h a p t e n i c w i t h r e s p e c t t o n a t i v e l y s o z y m e a n d i t s a n t i s e r u m , and i n c l u d e d t h e N H ^ - t e r m i n a l p o r t i o n o f t h e " l o o p p e p t i d e " s t u d i e d by A r n o n and S e l a ( 1 9 6 9 ) . T h e r e s u l t s r e p o r t e d h e r e i n d i c a t e t h a t t h e r e a r e a r e a s o f a n t i g e n i c i t y on C_. p a s t e u r i a n u m f e r r e d o x i n w h i c h do n o t d i r e c t l y i n v o l v e p a r t i c i p a t i o n o f c y s t e i n e r e s i d u e s , and t h a t t h e C O O H - t e r m i n a l o c t a p e p t i d e i s one o f t h e s e . I t w o u l d be i n t e r e s t i n g t o d e t e r m i n e t h e s p e c i e s a g a i n s t w h i c h a n t i b o d i e s a r e p r o d u c e d when n a t i v e f e r r e d o x i n i s u s e d a s an a n t i g e n , and f u r t h e r s t u d i e s w i l l i n v o l v e t e s t i n g o f o t h e r a r e a s o f t h e m o l e c u l e f o r t h e i r a b i l i t y t o b i n d a n t i b o d y d i r e c t e d a g a i n s t t h e w h o l e m o l e c u l e , and t o e s t a b l i s h t h e minimum s e q u e n c e o f a m i n o a c i d s w i t h i n t h e C O O H - t e r m i n a l o c t a p e p t i d e w h i c h i s c a p a b l e o f b i n d i n g a n t i b o d y d i r e c t e d a g a i n s t t h e w h o l e m o l e c u l e . CONCLUDING REMARKS T h e w o r k d e s c r i b e d i n t h i s t h e s i s i n v o l v e d a s t u d y o f t h e a n t i g e n i c c h a r a c t e r i s t i c s o f t h e e l e c t r o n t r a n s p o r t p r o t e i n , f e r r e d o x i n i s o l a t e d f r o m C l o s t r i d i u m p a s t e u r i a n u m . F o u r m a i n c o n c l u s i o n s c a n be d r a w n f r o m t h e d a t a c o m p i l e d i n t h i s t h e s i s : ( 1 ) F e r r e d o x i n and i t s p e r f o r m i c a c i d o x i d i z e d d e r i v a t i v e a r e a n t i g e n i c i n r a b b i t s a s m e a s u r e d by t h e c o m p l e m e n t f i x a t i o n t e s t . T h e o x i d i z e d d e r i v a t i v e a p p e a r s t o be a b e t t e r a n t i g e n and e v o k e s t h e p r o d u c t i o n o f p r e c i p i t a t i n g a n t i b o d y . T h i s c o n c l u s i o n , h o w e v e r , i s t e n t a t i v e , s i n c e o n l y o n e a n i m a l was u s e d f o r e a c h i m m u n i z a t i o n , tn t h e c a s e o f p e r f o r m i c a c i d o x i d i z e d f e r r e d o x i n a n t i s e r u m , c r o s s -r e a c t i o n was a l s o o b s e r v e d by t h e q u a n t i t a t i v e p r e c i p i t a t i o n m e t h o d . (2) C y s t e i n e i s n o t c r i t i c a l l y i n v o l v e d i n t h e a m i n o a c i d s e q u e n c e s o f at l e a s t two o f t h e a n t i g e n i c r e g i o n s o f t h e f e r r e d o x i n m o l e c u l e , s i n c e c r o s s - r e a c t i o n , a s m e a s u r e d by c o m p l e m e n t f i x a t i o n , was o b s e r v e d i n a l l i n s t a n c e s b e t w e e n n a t i v e f e r r e d o x i n a n t i s e r u m and p e r f o r m i c a c i d o x i d i z e d f e r r e d o x i n a n t i s e r u m w i t h f o u r f o r m s o f f e r r e d o x i n . E a c h f o r m c o n t a i n e d d i f f e r e n t c y s t e i n e r e s i d u e s ; n a t i v e f e r r e d o x i n , a p o f e r r e d o x i n , p e r f o r m i c a c i d o x i d i z e d f e r r e d o x i n and c a r b o x y a m i d o -m e t h y l a t e d f e r r e d o x i n . (3) A n t i b o d i e s p r o d u c e d by i n o c u l a t i o n o f an a d j u v a n t p r e p a r a t i o n o f n a t i v e f e r r e d o x i n a p p e a r t o be d i r e c t e d a g a i n s t a m o d i f i e d s p e c i e s o f t h e p r o t e i n . F e r r e d o x i n i s p r o b a b l y d e n a t u r e d a n d f r e e d o f l i g a n d s i n v i v o b e f o r e s t i m u l a t i n g an a n t i b o d y r e s p o n s e , s i n c e t h e a n t i s e r u m p r o d u c e d r e a c t e d t o a g r e a t e r d e g r e e w i t h a p o f e r r e d o x i n a n d c a r b o x a m i d p m e t h y l a t e d f e r r e d o x i n t h a n w i t h i t s s u p p o s e d l y h o m o l o g o u s a n t i g e n , a s m e a s u r e d by c o m p l e m e n t f i x a t i o n . B o t h a p o f e r r e d o x i n and c a r b o x a m i d o m e t h y l a t e d f e r r e d o x i n a r e d e n a t u r e d and f r e e o f i r o n and h y d r o g e n s u l f i d e . (4) The C O O H - t e r m i n a l o c t a p e p t i d e i s an a r e a o f a n t i g e n i c i t y i n b o t h . n a t i v e f e r r e d o x i n and i t s p e r f o r m i c a c i d o x i d i z e d d e r i v a t i v e a s m e a s u r e d by i n h i b i t i o n o f c o m p l e m e n t f i x a t i o n a n d p r e c i p i t a t i o n , and by d i r e c t b i n d i n g s t u d i e s u s i n g t h e e q u i l i b r i u m d i a l y s i s m e t h o d . 70 LITERATURE CITED 1. A r n o n , R . , a n d M . S e l a . 1969. A n t i b o d i e s t o a u n i q u e r e g i o n i n l y s o z y m e p r o v o k e d by a s y n t h e t i c a n t i g e n c o n j u g a t e . P r o c . N a t . A c a d . S c i . 62:163-170. 2. A r n o n , R . , M. S e l a , A . Y a r o n , a n d M . A . S o b e r . 1965. P o l y -l y s i n e - s p e c i f i c a n t i b o d i e s a n d t h e i r r e a c t i o n w i t h o l f g o l y s i n e s . B i o c h e m . 41:948~953-3. A t a s s i , M . Z . 1967. P e r i o d a t e o x i d a t i o n o f s p e r m w h a l e m y o g l o b i n and t h e r o l e o f t h e m e t h i o n i n e r e s i d u e s i n t h e a n t i b o d y r e a c t i o n . B i o c h e m . J . 102:478-487. 4. A t a s s i , M . Z . 1967- S p e c i f i c c l e a v a g e o f t r y p t o p h y l p e p t i d e b o n d s w i t h p e r i o d a t e i n s p e r m w h a l e m y o g l o b i n . A r c h . B i o c h e m . B i o p h y s . 120:56-59. 5. A t a s s i , M . Z . 1968. I m m u n o c h e m i s t r y o f s p e r m w h a l e m y o g l o b i n . I I I . M o d i f i c a t i o n o f t h e t h r e e t y r o s i n e r e s i d u e s and t h e i r r o l e s i n t h e a n t i g e n i c r e a c t i v i t y . B i o c h e m . 7^ :3078-3084. 6. A t a s s i , M . Z . a n d D . R . C a r u s o . 1968. I m m u n o c h e m i s t r y o f s p e r m w h a l e m y o g l o b i n . I I . 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