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The antigenicity of human and dog carboxy-hemoglobin preparations
408
SI-IOI~T
COMMOMCATIONS sc
23oo3
The antigenicity of human and do 9 earboxy-hemoglobin preparations The ability of h u m a n hemoglobin to elicit antibody formation in the rabbit has been established ~-3, but there is relatively little quantitative data available on the amount and heterogeneity of the antibody elicited. The present study is concerned with the antigenicity and cross-reactivity of both human and dog hemoglobin, the heterogeneity of the antibody response to the preparations used, and the cross-reactivity between antisera to hemoglobin and various synthetic polypeptide antigens. Both human and dog hemoglobin were prepared b y the same method 4 employing blood from a single human (0 positive) donor and blood from a single dog. The blood was centrifuged for I h at 3000 x g and then the serum and huffy coat were removed. The cells were washed thrice with 3 times their volume of 0. 9 % NaC1 and once with 3 times their volume of 1.2 % NaC1; following each wash the cells were separated b y centrifugation at 12000 × g for I h. The cells were then disrupted b y adding an equal volume of water and freezing and thawing twice. The resultant hemoglobin solution was mixed with washed, shredded asbestos to adsorb any particulate m a t t e r and the asbestos was removed b y centrifugation at 12 ooo × g for I h; this procedure was repeated three times. The final hemoglobin solution was obtained from the u p p e r HUMAN Hb-ANTI-HUMAN Hb
A
DOGHb-ANTI-HUMAN Hb(II)
:3-
B
~
~ o z
I DOG Hb-ANTI-DOG Hb
II
I C
P H HUMANHb-ANTI-DOG Hb(II)
I D
"(3 < 20O
I00
Oo
/
I Ioo
.
--X
I 400 o Antigen N (pg/rnl)
I Ioo
II
I 400
Fig. I. T h e precipitin c u r v e s on t h e left (A, C) s h o w t h e h o m o l o g o u s r e a c t i o n b e t w e e n t h e pooled a n t i - h e m o g l o b i n sera f r o m t h e different bleedings (I, II, III) a n d t h e i r respective antigens. T h e precipitin c u r v e s on t h e r i g h t (B, D) s h o w t h e cross-reactions b e t w e e n dog a n d h u m a n h e m o g l o b i n a n d t h e a n t i s e r a to h u m a n a n d dog hemoglobin, respectively.
Biochim. Biophys. dcla, 86 (1964) 4 o 8 - 4 1 o
SHORT COMMUNICATIONS
40~
two-thirds of the supernatant from the last treatment with asbestos, it was reacted. with carbon monoxide and stored frozen until used. One group of New Zealand white rabbits weighing 2- 3 kg was immunized with human carboxy-hemogtobin and another group, with dog carboxy-hemoglobin according to the following schedule: io mg of antigen in Freund's complete adjuvant were injected subcutaneously and 3 weeks later Io mg in o.I 5 M saline-phosphate buffer (pH 7.6) were injected subcutaneously; after 3 weeks three intravenous ino. jections of IO mg each were given on alternate days. One week after the last intravenous injection the animals were bled (Bleeding I). The second course of immunization started I week later and consisted of two intravenous injections of Io mg each on alternate days; the animals were bled z week after the last injection (Bleeding II), Two days later the animals were exsanguinated (Bleeding III). Each of the 14 rabbits immunized with human hemoglobin and each of the Ix animals immunized with dog hemoglobin made antibody. Equal aliquots of sera from each bleeding were pooled (Antisera I, II, I I I ) and used for the precipitin curves with carboxy-hemoglobin as the antigen. In order to demonstrate that the carboxy-hemoglobin was not altered b y incorporating it into Freund's adjuvant, it was extracted with saline-phosphate buffer from an aliquot of the immunizing mixture and the spectrum was taken on a Beckman DK-2A recording speetrophotometer. There was no difference between the spectrum of the carboxy-hemoglobin before and after incorporation into Freund's adjuvant. The addition of Na2S~O 4 to the extracted carboxy-hemoglobin failed to bring out any absorption bands indicative of an alteration in the hemoglobin structure. Dog hemoglobin elicited more antibody than h u m a n hemoglobin. The precipitin curves for both the human and dog hemoglobin preparations are shown in Fig. x, Their shapes are complex and this indicates the presence of several antigenic systems. The contaminating antigens m a y be minor hemoglobin components or other porphyrincontaining proteins, at least to a large extent, since the precipitates dissolved in o.~5 lVf acetic acid all contained material with the characteristic porphyrin absorption band at 4x6 m/~. The human and dog hemoglobin preparations cross-react to the same extent (zo-3o %) with each other's antiserum. The precipitin curves for these cross-reactions are shown in Fig. x. Since antisera to the synthetic polypeptides Glu56Ly%sTyr6 and Olu~lLysa3Tyr16 cross-react with human hemoglobin ~, it would be of interest to know whether the synthetic polypeptides cross-react with antisera to hemoglobin. In order to study this possibility, separate aliquots of pooled antisera from each of the three bleedings from rabbits immunized with human and dog hemoglobin, respectively, were mixed with varying amounts of Glu56Lys3sTyr6 or Glu51Lys3~Tyr16. The mixtures were incubated for 5 days at 4 ° and then analyzed in the usual manner for the presence of immune precipitateS: none was found in any of the various mixtures. Thus the two synthetic polypepiides can not cross-react with anti-human or anti-dog hemoglobin sera containing antibody (xoo-3oo ~ [ N/rely, despite the fact that hemoglobin can cross-react with anti-synthetic polypeptide sera containing similar amounts of antibody. From these results and from our previous findings 5,6 it appears that antibodies to the less structurally specific synthetic polypeptides can cross-react with many" Biochirn. BioC~hvs. dora.
86 (~o64) ao8-aio
410
SHORT COMMUNICATIONS
proteins, but that antibodies directed against the highly specific antigenic sites on proteins can not cross-react with synthetic polypeptides. In summary, both h u m a n and dog hemoglobin are antigenic in the rabbit and the latter is the better antigen. The hemoglobins cross-react to the same extent (2o-3 ° %) with each other's antiserum. The preparations studied also contain other antigens which are presumably minor hemoglobin components or other porphyrincontaining proteins. Antisera to human and dog hemoglobin fail to cross-react with two synthetic polypeptides, despite the fact that the antisera to the polypeptides cross-react with hemoglobin. One of the authors (T. J.G.) is recipient of a Lederle Medical Faculty Award (1962-1965). This study was supported b y a grant from the National Heart Institute (H-I77I).
Laboratory of Chemical Pathology, Department of Pathology, Harvard Medical School, Boston, Mass. (U.S.A.) A. 2 M. 3 G. 4 G. 5 T. 6 H. 1
HEINZ W. KUNZ TI-IOMAS J. GILL U I
I. CI~ER~IOrF, Blood, 8 (1953) 413 • GOODMAN AND D. H. CAMPBELL, Blood, 8 (1953) 422. 1:~. E. ~TAYLOR AND M. E. ADAIR, f . Immunol., 88 (1962) 4Ol. H. BEAVEN, 1V[. J. ELLIS AND J. C. WHITE, Brit. J. Haematol., 6 (196o) I. J. GILL I I I AND L. S. MATHEWS, J. Biol. Chem., 238 (1963) 1373. W. KUNZ AND T. J. GILL III, Biochim. Biophys. Acta, in the press.
Received December i6th, 1963 Biochim. Biophys. Acta, 86 (1964) 4o8-41o
sc 23 o12
Group-characteristic differences in amino acid composition between Bence-Jones proteins of Burtin's Types I and I I Even before Bence-Jones proteins had been isolated in a state of purity satisfying the usual tests of homogeneity, evidence had been gathered suggesting that no two Bence-Jones proteins would be identical 1. Recently we described 2-~ the isolation of five Bence-Jones proteins homogeneous in starch-gel electrophoresis. Four belonged to Burtin's Type I, and one to Burtin's Type II. No two of them appeared to have identical amino acid compositions, while the difference between the protein of Type I I on the one hand and the four proteins of Type I on the other appeared to be no more pronounced than the mutual differences between the proteins of Type I. We have now purified, along similar lines, another four proteins, three (Nos. IOO, I:E9 and 12o) of Type I I and one (No. 121) of Type I. They, too, are homogeneous in starch-gel electrophoresis. The results of the amino acid analyses of these proteins, together with the results already published on the proteins No. IOI, lO9, IiO, I I I and 112 are assembled in Table I. I t is evident that significant differences exist between the two types, which could not be detected as long as only one specimen of Type I I was available. The methionine content of the Type I I proteins was always nil, whereas in Type I, I or 2 residues were found per 45 ooo. Furthermore the contents of phenylalanine Biochim. Biophys. Acta, 86 (1964) 41o-412
SI-IOI~T
COMMOMCATIONS sc
23oo3
The antigenicity of human and do 9 earboxy-hemoglobin preparations The ability of h u m a n hemoglobin to elicit antibody formation in the rabbit has been established ~-3, but there is relatively little quantitative data available on the amount and heterogeneity of the antibody elicited. The present study is concerned with the antigenicity and cross-reactivity of both human and dog hemoglobin, the heterogeneity of the antibody response to the preparations used, and the cross-reactivity between antisera to hemoglobin and various synthetic polypeptide antigens. Both human and dog hemoglobin were prepared b y the same method 4 employing blood from a single human (0 positive) donor and blood from a single dog. The blood was centrifuged for I h at 3000 x g and then the serum and huffy coat were removed. The cells were washed thrice with 3 times their volume of 0. 9 % NaC1 and once with 3 times their volume of 1.2 % NaC1; following each wash the cells were separated b y centrifugation at 12000 × g for I h. The cells were then disrupted b y adding an equal volume of water and freezing and thawing twice. The resultant hemoglobin solution was mixed with washed, shredded asbestos to adsorb any particulate m a t t e r and the asbestos was removed b y centrifugation at 12 ooo × g for I h; this procedure was repeated three times. The final hemoglobin solution was obtained from the u p p e r HUMAN Hb-ANTI-HUMAN Hb
A
DOGHb-ANTI-HUMAN Hb(II)
:3-
B
~
~ o z
I DOG Hb-ANTI-DOG Hb
II
I C
P H HUMANHb-ANTI-DOG Hb(II)
I D
"(3 < 20O
I00
Oo
/
I Ioo
.
--X
I 400 o Antigen N (pg/rnl)
I Ioo
II
I 400
Fig. I. T h e precipitin c u r v e s on t h e left (A, C) s h o w t h e h o m o l o g o u s r e a c t i o n b e t w e e n t h e pooled a n t i - h e m o g l o b i n sera f r o m t h e different bleedings (I, II, III) a n d t h e i r respective antigens. T h e precipitin c u r v e s on t h e r i g h t (B, D) s h o w t h e cross-reactions b e t w e e n dog a n d h u m a n h e m o g l o b i n a n d t h e a n t i s e r a to h u m a n a n d dog hemoglobin, respectively.
Biochim. Biophys. dcla, 86 (1964) 4 o 8 - 4 1 o
SHORT COMMUNICATIONS
40~
two-thirds of the supernatant from the last treatment with asbestos, it was reacted. with carbon monoxide and stored frozen until used. One group of New Zealand white rabbits weighing 2- 3 kg was immunized with human carboxy-hemogtobin and another group, with dog carboxy-hemoglobin according to the following schedule: io mg of antigen in Freund's complete adjuvant were injected subcutaneously and 3 weeks later Io mg in o.I 5 M saline-phosphate buffer (pH 7.6) were injected subcutaneously; after 3 weeks three intravenous ino. jections of IO mg each were given on alternate days. One week after the last intravenous injection the animals were bled (Bleeding I). The second course of immunization started I week later and consisted of two intravenous injections of Io mg each on alternate days; the animals were bled z week after the last injection (Bleeding II), Two days later the animals were exsanguinated (Bleeding III). Each of the 14 rabbits immunized with human hemoglobin and each of the Ix animals immunized with dog hemoglobin made antibody. Equal aliquots of sera from each bleeding were pooled (Antisera I, II, I I I ) and used for the precipitin curves with carboxy-hemoglobin as the antigen. In order to demonstrate that the carboxy-hemoglobin was not altered b y incorporating it into Freund's adjuvant, it was extracted with saline-phosphate buffer from an aliquot of the immunizing mixture and the spectrum was taken on a Beckman DK-2A recording speetrophotometer. There was no difference between the spectrum of the carboxy-hemoglobin before and after incorporation into Freund's adjuvant. The addition of Na2S~O 4 to the extracted carboxy-hemoglobin failed to bring out any absorption bands indicative of an alteration in the hemoglobin structure. Dog hemoglobin elicited more antibody than h u m a n hemoglobin. The precipitin curves for both the human and dog hemoglobin preparations are shown in Fig. x, Their shapes are complex and this indicates the presence of several antigenic systems. The contaminating antigens m a y be minor hemoglobin components or other porphyrincontaining proteins, at least to a large extent, since the precipitates dissolved in o.~5 lVf acetic acid all contained material with the characteristic porphyrin absorption band at 4x6 m/~. The human and dog hemoglobin preparations cross-react to the same extent (zo-3o %) with each other's antiserum. The precipitin curves for these cross-reactions are shown in Fig. x. Since antisera to the synthetic polypeptides Glu56Ly%sTyr6 and Olu~lLysa3Tyr16 cross-react with human hemoglobin ~, it would be of interest to know whether the synthetic polypeptides cross-react with antisera to hemoglobin. In order to study this possibility, separate aliquots of pooled antisera from each of the three bleedings from rabbits immunized with human and dog hemoglobin, respectively, were mixed with varying amounts of Glu56Lys3sTyr6 or Glu51Lys3~Tyr16. The mixtures were incubated for 5 days at 4 ° and then analyzed in the usual manner for the presence of immune precipitateS: none was found in any of the various mixtures. Thus the two synthetic polypepiides can not cross-react with anti-human or anti-dog hemoglobin sera containing antibody (xoo-3oo ~ [ N/rely, despite the fact that hemoglobin can cross-react with anti-synthetic polypeptide sera containing similar amounts of antibody. From these results and from our previous findings 5,6 it appears that antibodies to the less structurally specific synthetic polypeptides can cross-react with many" Biochirn. BioC~hvs. dora.
86 (~o64) ao8-aio
410
SHORT COMMUNICATIONS
proteins, but that antibodies directed against the highly specific antigenic sites on proteins can not cross-react with synthetic polypeptides. In summary, both h u m a n and dog hemoglobin are antigenic in the rabbit and the latter is the better antigen. The hemoglobins cross-react to the same extent (2o-3 ° %) with each other's antiserum. The preparations studied also contain other antigens which are presumably minor hemoglobin components or other porphyrincontaining proteins. Antisera to human and dog hemoglobin fail to cross-react with two synthetic polypeptides, despite the fact that the antisera to the polypeptides cross-react with hemoglobin. One of the authors (T. J.G.) is recipient of a Lederle Medical Faculty Award (1962-1965). This study was supported b y a grant from the National Heart Institute (H-I77I).
Laboratory of Chemical Pathology, Department of Pathology, Harvard Medical School, Boston, Mass. (U.S.A.) A. 2 M. 3 G. 4 G. 5 T. 6 H. 1
HEINZ W. KUNZ TI-IOMAS J. GILL U I
I. CI~ER~IOrF, Blood, 8 (1953) 413 • GOODMAN AND D. H. CAMPBELL, Blood, 8 (1953) 422. 1:~. E. ~TAYLOR AND M. E. ADAIR, f . Immunol., 88 (1962) 4Ol. H. BEAVEN, 1V[. J. ELLIS AND J. C. WHITE, Brit. J. Haematol., 6 (196o) I. J. GILL I I I AND L. S. MATHEWS, J. Biol. Chem., 238 (1963) 1373. W. KUNZ AND T. J. GILL III, Biochim. Biophys. Acta, in the press.
Received December i6th, 1963 Biochim. Biophys. Acta, 86 (1964) 4o8-41o
sc 23 o12
Group-characteristic differences in amino acid composition between Bence-Jones proteins of Burtin's Types I and I I Even before Bence-Jones proteins had been isolated in a state of purity satisfying the usual tests of homogeneity, evidence had been gathered suggesting that no two Bence-Jones proteins would be identical 1. Recently we described 2-~ the isolation of five Bence-Jones proteins homogeneous in starch-gel electrophoresis. Four belonged to Burtin's Type I, and one to Burtin's Type II. No two of them appeared to have identical amino acid compositions, while the difference between the protein of Type I I on the one hand and the four proteins of Type I on the other appeared to be no more pronounced than the mutual differences between the proteins of Type I. We have now purified, along similar lines, another four proteins, three (Nos. IOO, I:E9 and 12o) of Type I I and one (No. 121) of Type I. They, too, are homogeneous in starch-gel electrophoresis. The results of the amino acid analyses of these proteins, together with the results already published on the proteins No. IOI, lO9, IiO, I I I and 112 are assembled in Table I. I t is evident that significant differences exist between the two types, which could not be detected as long as only one specimen of Type I I was available. The methionine content of the Type I I proteins was always nil, whereas in Type I, I or 2 residues were found per 45 ooo. Furthermore the contents of phenylalanine Biochim. Biophys. Acta, 86 (1964) 41o-412