Antibodies against pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate

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BBA 25 617 ANTIBODIES AGAINST PYRIDOXAL 5'-PHOSPHATE AND PYRIDOXAMINE 5'-PHOSPHATE Ft~LIX C6RDOBA, CONCEPCION GONZfl~LEZ AND PABLO RIVERA Departamento de Bioqulmica, Facultad de Medicina, Universidad de Mdxico, M~xico (Mdxico)

(Received March Ist, 1966)

SUMMARY I. The antigenicity of pyridoxal 5'-phosphate in rabbits was tested by immunization of the animals with the coenzyme coupled to bovine serum albumin. The coupling of pyridoxal phosphate to albumin, as well as to other proteins, was achieved by reduction (at pH 7.5) with sodium borohydride. 2. The antiserum was precipitated specifically with pyridoxal phosphate-bovine serum albumin conjugate, with urease-pyridoxal phosphate, with ferritin-pyridoxal phosphate, with ribonuclease-pyridoxal phosphate and with rabbit y-globulinpyridoxal phosphate. 3. The precipitation of pyridoxal phosphate-rabbit v-globulin complex by the antiserum was inhibited, in increasing order, by previous incubation of the antibodies with HP042-, deoxypyridoxine, pyridine, pyridoxal, pyridoxal phosphate and pyridoxamine phosphate. The results suggest the formation of specific antibodies against the pyridoxal phosphate structure in the protein conjugate. The phosphate group and the C-N bond joining pyridoxal phosphate to bovine serum albumin appear to be important for the serological specificity. 4-The antiserum showed reactivity to other phosphorylated compounds like adenosine 5'-phosphate but did not inhibit or precipitate pig heart aspartate aminotransferase (EC 2.6.1.1).

INTRODUCTION It has been shown that enzymes containing pyridoxal 5'-phosphate are antigenic in animals and produce antibodies capable of precipitation and inhibition of the enzymes 1, 2. Antibodies to mammalian aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) do not appear to be formed, however, against the enzymic site, where the cofactor is located. Other antigenic groups in the enzyme seem better suited to elicit antibodies and to modify the enzymic behavior because of their specific combination 3. In order to find out if pyridoxal 5'-phosphate could become antigenic we decided to prepare compounds in which pyridoxal 5'-phosphate was coupled to nonenzymic proteins and to investigate if antibodies were not directed to the pyridoxal 5'-phosphate moiety, as it appears to be in some pyridoxal 5'-phosphate enzymes. Biochim. Biophys. Acta, 127 (I966/ IM-t58

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Binding of pyridoxal 5'-phosphate to enzymes can be stabilized by a reductive reaction with sodium borohydride. This procedure, described by FISCHER et al. 4, for the elucidation of the amino acids at the active site of phosphorylase (EC 2.4.i.i ) and of aspartate aminotransferase ~, was recently used by CHURCHICH6 for demonstrating energy transfer to pyridoxal 5'-phosphate molecules reductively bound to human serum albumin. By using pyridoxal 5'-phosphate and bovine serum albumin we have been able to prepare an antigen that, upon iniection into rabbits, produced an antiserum reactive with different pyridoxal 5'-phosphate-protein conjugates. The antigenicity appears to reside in the phosphate chain of pyridoxat 5'-phosphate and, to a lesser degree, in the stable C--N linkage joining pyridoxal 5'-phosphate to the protein molecule. METHODS

Conjugation of pyridoxal 5'phosphate to bovine serum albumin was done essentially as described by CHURCmCH6. Bovine serum albumin (300 rag) (Armour and Co., U.S.A.) was dissolved in 12o ml of 0.05 M phosphate buffer (pH 7.5), and 12o mg of pyridoxal 5'-phosphate (Nutritional Biochemicals Corporation, U.S.A.) was slowly added with gentle stirring at a temperature of 37 °. The pH was maintained at 7.5 with NaOH. After 30 rain the mixture was chilled in an ice-bath and 30 mg of sodium borohydride were added. The stirring was continued and the reduction allowed to take effect during 15 rain. The reaction was stopped with glacial acetic acid and the solution enclosed in Visking tubing and dialyzed for 48 h in the cold, against several 4-1 portions of 0.9 °"o NaC1 solution. Dialysis was prolonged until the absorbance of the diffusate, at 317 rot* for pyridoxal 5'-phosphate 7, was negligible. As a further test for free pyridoxal 5'-phosphate an aliquot of the protein solution was precipitated with IO % trichloroacetic acid and the supernatant assayed as before. Crystalline (2 71) horse spleen ferritin (Nutritional Biochemicals Corporation), urease Special Purity Type V (Sigma Co., U.S.A.), crystallized (5 " ) bovine pancreas ribonuclease (Sigma) and rabbit v-globulin (immunoglobulin (;), isolated by sodium sulfate precipitation and purified by starch-block electrophoresis from pooled normal rabbit serum, were coupled to pyridoxal 5'-phosphate (at pH 7.5) using the identical procedure as described for the conjugation to bovine serum albumin. Pyridoxal 5'-phosphate substitution was assessed by spectrophotometric scanning, in the quartz cuvettes, using a Zeiss P M Q I I spectrophotometer at a wavelength of 325 m/~ and a molar absorption coefficient of 83oo, for pyridoxamine 5'-phosphate ~. The ultraviolet spectra of the solutions, in the range from 24o to 35o m~, were obtained in the same instrument. The amount of pyridoxal 5'-phosphate, in the pyridoxal 5'-phosphate-ferritin preparations, was estimated b y direct phosphate analysis using the technique of Fiske--SubbaRow, as in ref. 8. Molar pyridoxal 5'-phosphate labeling was calculated using molecular weight values of 465ooo for horse spleen ferritin 9, 48oooo for urease ~°, 15oooo for rabbit ~,-globulin ~t, 67ooo for bovine serum albumin ~2 and 127oo for bovine pancreas ribonuclease la. Protein was estimated with the biuret reagent and by the nesslerization procedure described b y LANNI, DILLON AND BEARD14. Bioch{m. Biophys. Acta, E27 (1906) 151-158

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Local strain female adult rabbits were immunized by subcutaneous administration of 50 mg of pyridoxal 5'-phosphate-bovine serum albumin mixed with Freund's adjuvants. A booster series started one month after the initial dose with, daily I ml, intradermic and intravenous injections of 5 mg/ml, of the same antigen suspended in alum. This procedure was repeated during 45 days. The animals were bled from the ear veins, the serum was separated from the clot and stored in the freezer, at --20 °, until needed. Immunoprecipitation of the pyridoxal 5'-phosphate-protein conjugates with the antisera was assayed in 2 % agar gel plates using special agar Noble (Difco) in 0. 9 % NaC1 solution. Diffusion occurred in a humid chamber, at room temperature for several days. Pure merthiolate (Lilly) in a I : IOOOOfinal dilution, was incorporated in the agar gel. Quantitative precipitin tests were set up with pyridoxal 5'-phosphate-ferritin, pyridoxal 5'-phosphate-urease and pyridoxal 5'-phosphate-rabbit ~,-globulin, against anti-pyridoxal 5'-phosphate-bovine serum albumin serum, in test tubes containing i ml of antiserum and 0.5 ml dilutions of each pyridoxal 5'phosphate-protein conjugate (because of the discrete precipitation obtained with pyridoxal 5'-phosphate-ribonuclease, this preparation was not used for this test). The tubes were stored in the cold chest for one week and the precipitates were spun down, washed three times with chilled saline and analyzed for protein by the Nessler procedure 14. Inhibition of precipitation was performed by mixing and storing in the cold I-ml volumes of the antisera with different concentrations each of pyridoxal 5'-phosphate, pyridoxamine phosphate, pyridoxal.HC1, pyridine, deoxypyridoxine, adenosine triphosphate and Na,HPO 4 dissolved in 0.05 M borate buffer (pH 8.5). After 24 h a calibrated amount of pyridoxal 5'-phosphate-9,-globulin, known to precipitate most of the antibodies to pyridoxal 5'-phosphate, was pipetted, and the tubes were replaced in the cold chest to attain final precipitation. The specific precipitates were isolated, washed and analyzed as before. RESULTS

The ultraviolet spectra of the various pyridoxal 5'-phosphate-protein conjugates employed in this work are displayed in Fig. I. The preparations were exhaustively dialyzed, and the protein concentration adjusted to about o.I nag protein per ml. The peak at 325-327 mtz corresponds to the one for free pyridoxamine 5'-phosphate at this pHe. The absorption band about 280 m/z, due to aromatic amino acids, appeared displaced.This shift was also noticed m the spectra of reduced non-pyrld0xal 5'-phosphate-substituted proteins, suggesting additional alterations in the molecule brought about b y the reductive treatment (see ref. 13). Because of the strong interference of the heme groups in ferritin the pyridoxal 5'-phosphate conjugate spectrum failed to show the characteristic band at 325 m/z (Fig. I). Pyridoxal 5'-phosphate substitution was verified by direct phosphate analysis. The extent of pyridoxal 5'-phosphate conjugation varied over wide ranges in the various proteins; 5 pyridoxal 5'-phosphate molecules were incorporated into bovine serum albumin (I mole of protein), 7 in rabbit y-globulin, 64 in urease and about 95 in ferritin. Ribonuclease showed the least extent of substitution with only 2 moles of pyridoxal 5'-phosphate per mole of protein. Biochim. Biophys. Acta, 127 (1966) 151-158

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Fig. 2 shows the precipitation bands of the anti-pyridoxal 5'-phosphate-bovine serum albumin serum with the pyridoxal 5'-phosphate-protein derivatives, in get diffusion plates. Bovine serum albumin and pyridoxal 5'-phosphate-bovine serum albumin gave stronger precipitates than identical concentrations of the conjugates. This is due to the precipitation of bovine serum albumin antibodies formed during the

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280 300 320 340 360 Wovelength (rap) Fig. I. Ultraviolet spectra of pyridoxal 5 ' - p h o s p h a t e - p r o t e i n conjugates dissolved in 0.9 °,o NaCI solution. - . . . . . . , pyridoxal phosphate--ferritin; . . . . . , pyridoxal p h o s p h a t e - b o v i n e s e r u m albumin, - . . . . , pyridoxal p h o s p h a t e - r i b o n u c l e a s e ; . . . . , pyridoxal p h o s p h a t e - u r e a s e ; pyridoxal p h o s p h a t e r a b b i t v-globulin; - - , bovine serum albumin. The concentration used was o.i m g / m l protein, with the exception of bovine s e r u m a l b u m i n which was o.o 7 mg/ml.

Fig. 2. Precipitation of pyridoxal 5 ' - p h o s p h a t e - p r o t e i n conjugates by a n t i s e r u m against pyridoxal p h o s p h a t e - b o v i n e s e r u m albumin, in gel diffusion plates. AP, pyridoxal p h o s p h a t e - b o v i n e s e r u m a l b u m i n ; G, pyridoxal p h o s p h a t e - r a b b i t T-globulin; A, bovine serum a l b u m i n ; F, pyridoxal p h o s p h a t e - f e r r i t i n ; R, pyridoxal p h o s p h a t e - r i b o n u c l e a s e ; and V, pyridoxal p h o s p h a t e ~urease. Center well: r a b b i t anti-pyridoxal p h o s p h a t e - b o v i n e s e r u m albunfin serum.

Biochim. t3iophys, dcta, 127 (I966j 15~-I58

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Pyridoxal 5'-phosphate-ferritin, pyridoxal 5'-phosphate-urease and pyridoxal 5'-phosphate-y-globulin produced distinct, though weaker, bands of precipitation. Pyridoxal 5'-phosphate-ribonuclease at high concentrations displayed a very fine precipitation band not visible in the photographic reproduction of Fig. 2. The fusion of pyridoxal 5'-phosphate-urease precipitate bands with those of pyridoxal 5'-phosphate-bovine serum albumin is clearly visible. Pyridoxal 5'-phosphate-ferritin precipitate on the other hand, crosses over the precipitate formed between bovine serum albumin and the antiserum, at the opposite side of the same plate. When bovine serum albumin was used to absorb the antiserum, similar precipitation bands (with respect to cross-reacting pyridoxal 5'-phosphate conjugates) were observed. Results of the quantitative precipitin tests, with the three best precipitating pyridoxal 5'-phosphate-proteins, are shown in Fig. 3- The precipitin curves obtained are similar to those reported for other immunological systems 15. The equivalence zone is a broad one, more noticeable with pyridoxal 5'-phosphate-ferritin, and the inhibition takes place in an ample range of antigen excess.

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Phosphete in conjugQte(lJg) Fig. 3. Quantitative precipitation curves of pyridoxal phosphate-protein conjugates against rabbit, anti-pyridoxal p h o s p h a t e - b o v i n e serum albumin serum. O - - Q , pyridoxal p h o s p h a t e ferritin; O - - O , pyridoxal p h o s p h a t e - u r e a s e ; [ ] - - Q , pyridoxal p h o s p h a t e - r a b b i t y-globulin. The amounts of cross-reacting antigens used were plotted in abscissa as/~g of phosphate in the conjugates.

Pyridoxal 5'-phosphate-ferritin conjugate, richer in pyridoxal 5'-phosphate groups, precipitated more antibody than either of the other two conjugates tested but this characteristic by itself is not sufficient to explain the difference in precipitation; pyridoxal 5'-phosphate-urease and pyridoxal 5'-phosphate-y-globulin differed, in this respect, by a factor of 9, and the amount of antibody precipitated by the two preparations was similar (Fig. 3). Finally, Fig. 4 shows the results of the experiments on the inhibition of precipitation with pyridoxal 5'-phosphate and related chemical substances. The precipitating Biochim. Biophys. Acta, 127 (I966) 151-158

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system employed was anti-pyridoxal 5'-phosphate-bovine serum albumin serum and rabbit y-globulin-pyridoxal 5'-phosphate, in amounts near the maximum of precipitation. A solution of 3 mM pyridoxal 5'-phosphate (see METHODS)reduced precipitation by 50 %. Pyridoxamine phosphate, nevertheless, is still a better inhibitory substance than the hapten itself; a solution o.2 mM is equally effective in reducing the precipitation to 5o % , as compared with the controls. Very low inhibition of precipitation resulted with pyridine; 25 % inhibition with a o.3 M concentration range.

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Fig. 4. Results of the inhibition of precipitation with pyridoxal phosphate rabbit ?~-globulin and serum anti-pyridoxal phosphate-bovine serum albumin, using free pyridoxal phosphate and other haptenie compounds. O--O, pyridoxal S-phosphate; O O, pyridoxamine S-phosphate; [] ~, deoxypyridoxine; I - - I , pyridoxal; G G, pyridine; , - - A , phosphate (HPO42); × - - x , ATP. The compounds tested were dissolved in o.o5 M borate buffer (pH 8.5) and added 24 h before the precipitating antigen. It is assumed, because of the above results, that antibodies to pyridoxal 5'phosphate with specificity not primarily for the pyridine ring (at least not for the ring core in pyridoxal 5'-phosphate) have been produced. When free pyridoxal and deoxypyridoxine were assayed as inhibitors, both were found to reduce precipitation much less than the phosphorylated derivatives tested previously. This finding was consistent with the interpretation already stated. Since the only common residue, both in pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate (aside from the substituted pyridine ring already tested with pyridoxal and found to give weak inhibition) was organic phosphate, it appeared that antibodies were directed mainly to this grouping in the two compounds. To test this idea ATP and inorganic phosphate, HPO42- (sodium) were similarly assayed for inhibition of precipitation. It was found that, regardless of the overall structural difference between pyridoxal 5'-phosphate and ATP, the last compound was indeed a fairly good inhibitory substance; a solution of about i i mM in ATP reduced precipitation to 5o % values. Inorganic phosphate, on the contrary, was found to behave as Biochim. Biophys. ,4cta, ~z 7 ~I966) 15I-I58

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a poor precipitation inhibitor--only 30 % with a concentration as high as 15o mM. The antiserum was assayed several times, at different pH values, in gel diffusion plates against purified aspartate-aminotransferase from pig heart. In no instance was a band of precipitation detected. In enzyme neutralization tests, previously described 3, the same enzyme was mixed with anti-pyridoxal 5'-phosphate-bovine serum albumin serum. Here, again, no inhibition of transamination was observed.

DISCUSSION

Some of the reactions of pyridoxal 5'-phosphate-protein conjugates with the antiserum displayed multiple precipitation bands. Pyridoxal 5'-phosphate-ferritin, in repeated experiments, precipitated as a single band, but pyridoxal 5'-phosphateurease gave 3 bands and pyridoxal 5'-phosphate-y-globulin 2 bands of precipitation. Pyridoxal 5'-phosphate-ribonuclease showed only one light band. Several explanations can be put forward in relation of this multiplicity of bands ; parallel lines of precipitation (instead of a unique one) could be due to the extra, complement(C')-dependent, precipitate band which appears in immunological systems employing antihapten antibodies 16. Separation of subunits, or even reductive breaking of peptide bonds by treatment with sodium borohydride (see ref. I3), gave rise to heterogeneity of the pyridoxal 5'-phosphate conjugates. This would probably produce a multiple precipitation pattern. The possibility that one of the extra bands was due to a non-specific reaction of the reduced protein, against antibodies to reduced protein, was ruled out because, when reduced non-pyridoxal 5'-phosphate-substituted proteins were reacted against the anti serum, they produced negative results. Inhibition of precipitation, with pyridoxal 5'-phosphate and pyridoxamine phosphate, allowed us to locate the antigenic points about the phosphate residue in the substituted pyridine ring structure. In this respect, ATP reactions were of interest because it suggested that antibodies formed with phosphate, in pyridoxal 5'-phosphate, are of a much broader spectrum than originally forecast. Since the extent of this reactivity to other phosphorylated compounds has not yet been fully explored (ADP and AMP were tried and found less inhibitory than either pyridoxal 5'-phosphate or ATP) it seems premature to speculate about it. Nevertheless, recent research with antibodies to nucleic acids 1~ appears to implicate, in some degree, reactions to the phosphate residues in the macromolecules. The same is valid with respect to phosphorylated polysaccharides in their reactions with specific antibodies TM. Inhibition induced by pyridoxal 5'-phosphate and pyridoxamine phosphate was markedly different. It was not possible to establish an explanation for these differences by the use of molecular models; however, these could still be due to a different type of phosphate binding, for antibody, in both compounds. Because pyridoxal 5'-phosphate-proteins, pyridoxal 5'-phosphate-dependent enzymes and pyridoxamine phosphate all contain a C-N linkage about the formyl imino (amino) group, a possible way to interpret the stronger inhibition with the aminated compound would be to assume that immunization with bovine serum albumin-pyridoxal 5'-phosphate had produced, in addition to antibodies for organic phosphate (see above), antibodies directed to the C-N linkage. Experiments using Biochim. Biophys. Acta, 127 (i966 ~ 151-158

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pyridoxal proteins as immunizing agents, instead of pyridoxal 5'-phosphate-proteins, currently under way in this laboratory may help to clear up this point. With regard to pyridoxal phosphate in enzymes, WADa AND MORINOva have stated that antibodies to aspartate aminotransferase are not binding to the enzymic site. Immunological studies with the same enzymes, from pig organs, have led CdRDOBAa to reach a similar conclusion. In view of the results presented here it can be concluded that pyridoxal 5'-phosphate is bound differently, at least in aspartate aminotransferase, than the way it binds to non-enzymic (non-pyridoxal 5'-phosphatedependent) proteins. This assumption receives strong support through the failure (>f pyridoxal 5'-phosphate antibodies to inhibit or precipitate the enzyme. The relevance of the phosphate moiety of pyridoxal 5'-phosphate for biological activity of transaminases has been reviewed recently bv BRAU~STEIN "°. The experiments reported here pointing to the immunological role of the phosphate group in pyridoxal 5'-phosphate-proteins have produced results which help to dispel a fear stated by the aforementioned author, He suspected that borohydride treatment ()f the enzymes would split off the phosphate from the lysyl--pyridoxal peptide recovered after reduction 5. Instead it seems that the failure to recover a phosphorylated derivative is due to a biologically important reason not yet fully understood. The pyridoxal phosphate bond is a very strong linkage, not split during the induction of phosphatespecific antibody formation. REFE RENCES I F. C6RDOBA, G. GONZ~,LEZ AND A. PEREZ, Nature, 194 (1962) I156. 2 N. VON LANG, intern. Arch. Allergy, 26 (1965) 345. 3 F. C6RDOBA, Syrup. hnmunol., Proe. intern. Congr. Pharmaeol. Therap., Sa~ Luis Polos[,

Mdxico, 1965, Excerpta Medica, Amsterdam, in the press. 4 E. H. FISCHER, A. B. KENT, E. R. SNYDER AND E. G. KREBS, J. Am. Chem. Soc., 80 (1958) 2900. 5 E. H. FISCHER, A. W. FORREY, J. L. HEDRICK, JR. C. HUGUES, A. B. KENT AND E. G. |(REBS, in E. E. SNELL, P. M. FASELLA, A. BRAUNSTEIN AND A. ROSSI-FANELLI, Chemical and Biological Aspects of Pyridoxal Catalysis, Macmillan, New York , N.Y., 1963, p. 5436 J. E. CHURCHICH, Biochim. Biophys. Acta, lO2 (i965) 28o. 7 D. E. METZLER AND E. E. SNELL, J. Am. Chem. Soc., 77 (I955) 243 I. 8 F. C. KOCH AND M. E. HANKE, Practical Methods in Biochemistry, Williams and \Vilkins, Baltimore, Md. 6th ed., 1953, p. 227. 9 P- M. HARRISON, Acta Cryst., 13 (196o) lO6O. IO M. DIXON AND E. C. WEBB, Enzymes, Longmans Green, London, 2nd ed., I904, p. 455. I I M. HE1DELBERGER AND K. O. PEDERSON, J. Exptl. Med., 65 (1937) 393. 12 G. 1. LOEB AND H. A. SCHERAGA, J. Phys. Chem., 60 (1956) 1633. 13 A. M. CRESTFIELD, S. MOORE AND W. I-[. STEIN, J. Biol. Chem., 238 (1963) 622. 14 F. LANNI, M. L. DILLON AND J. W. BEARD, Proe. Soe. Exptl. Biol. Med., 74 (I95°) 4. 15 E. A. KABAT AND M. MAYER, Experimental lmmunochemistry, Thomas, Springfield, t11.. 2ild ed., 1961, p. 5416 W. E. PAUL AND t3. BENACERRAF, J. lmmunol., 95 (1965) lO67. 17 B. F. ERLANGER AND S. M. BEISER, Proc. Natl. Acad. Sei. U.S., 52 (1964) ()S. 18 P. A. REBERS, E. HUR'WITZ AND M. HEIDELBERGER, J. Bacteriol., 82 (1961) 920. 19 H. WADA AND Y. MORINO, in R. S. HARRIS, I. G. WOOL AND J. A. LORAINE, Vitamins Hormo~es, Vol. 22, Academic Press, New Y o r k , N.Y., 1964, p. 411. 20 A. E. ]~RAUNSTEIN, in R. S. HARRIS, I. G. V~rOOL AND J. A. LORAINE, Vitamins Hormo~tes, Vol. 22, Academic Press, N e w York, N.Y. 1964, p. 45 I.

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