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C-reactive protein in the rabbit: Isolation, characterization and binding affinity to phosphocholine
lmraunochemistry, 1977,Vol.14, pp. 215-219. PergamonPress. Printedin Great Britain
C-REACTIVE PROTEIN IN THE RABBIT: ISOLATION, CHARACTERIZATION AND BINDING AFFINITY TO PHOSPHOCHOLINE* BRUCE ALLEN BACH,I" HENRY GEWURZ:~ and ALEXANDER P. OSMAND§ Department of Immunology, Rush Medical College Chicago, IL 60612, U.S.A. (Received 2 August 1976)
Abstract--Several structural and functional characteristics of rabbit C-reactive protein have been established. The protein has been isolated by affinity chromatography on pneumococcal C-polysaccharide linked to agarose. Following gel filtration, SDS-polyacrylamide gel electrophoresis indicated a subunit size of 22,900 +_ 400 daltons. Equilibrium gel filtration demonstrated a stoichiometric binding of (14C) phosphocholine with an association constant (K = 1.02 x 106M -1 at 5°C and 0.62 x 106M -~ at 15°C) in the medium range for antibody-hapten interactions. The amino acid composition for rabbit C-reactive protein is reported and compared with human C-reactive protein.
INTRODUCTION C-reactive protein (CRP) is a trace constituent of normal blood which is increased as much as 1000-fold during the acute phase of many inflammatory conditions. Additional studies in this laboratory have demonstrated the ability of CRP to fully activate the complement system (Osmand et al., 1975) and to inhibit certain lymphocyte (Mortensen et al., 1975; Mortensen & Gewurz, 1976) and platelet functions (Fiedel & Gewurz, 1976). Its unique characteristics include a calcium-dependent precipitation reaction with the pneumococcal C-polysaccharide (CPS), a cell wall teichoic acid of the pneumococcus. The specificity of the precipitation reaction of the human protein has been shown to be directed primarily towards the phosphocholine moiety (Volanakis & Kaplan, 1971). Early studies (Abernethy, 1937) in the search for experimental models for studying C-reactive protein described a C-reactive protein in the serum of monkeys with a type III pneumococcus infection. Anderson and McCarty (1951) described the presence of a C-reactive protein in rabbit serum which reacted and precipitated only with a less degraded form of C-polysaccharide, which they termed the Cx-polysaccharide. The rabbit protein was thus designated Cxreactive (CxRP). To date no further data have been presented concerning the apparent difference in reactivity between the human and rabbit proteins. Following the suggestion of Kushner and Somerville*These studies were supported by grants from the National Institutes of Health (AI 12870-02), the Leukemia Research Foundation, Inc., the Chicago Heart Association and the Hunter Trust. t Supported by a Boothroyd Medical Student Fellowship. Present address: Department of Pathology, Harvard Medical School, Boston, MA 02115. :~H.G. holds the Thomas J. Coogan, Sr. Chair in Immunology established by Marjorie Lindheimer Everett. § Address correspondence to: A. P. Osmand, Department of Immunology, Rush Medical College, Chicago, IL 60612, U.S.A. 215
Volanakis (.1973) the single term CRP will be used to refer to these proteins. This paper establishes similarities of rabbit to human CRP in terms of isolation by affinity chromatography, and binding specificity to the phosphocholine moiety of CPS as determined by equilibrium gel filtration. The extensive similarity in amino acid composition between rabbit and human CRP is also reported.
MATERIALS AND METHODS Rabbit C-reactive protein was prepared from acute phase sera essentially according to the method of Osmand et al. (1975). Sera were obtained from rabbits in the acute phase of inflammation 36-48 hr after multiple subcutaneous injections of a 1% croton oil suspension. Pooled sera were clarified by filtration through celite (Hyflo Supercel, Johns Mansville Products Corp.) and passed through an affinity column containing pneumococcal C-polysaccharide linked to Bio-Gel A-50m agarose beads (Bio Rad Laboratories). Polyacrylamide gel electrophoresis was performed exactly according to the method of Neville (1971). Gels stained with Coomassie brilliant blue according to the method of Weber & Osborn (1969) were analyzed densitometrically with a gel scanning device attached to a Beckman Acta C.V. spectrophotometer (Beckman Instruments). Binding affinities to (t4C) phosphocholine were determined by the method of Hummel & Dreyer (1962) with the modifications of Fairclough & Fruton (1966), and as redefined by Gotschlich & Edelman (1967). Binding affinity experiments were performed in jacketed columns (0.7 × 50 cm) of Sephadex (3-25 maintained at a constant temperature by a circulating water bath (Forma Scientific Co.). Radioactive (14C) phosphocholine was obtained from Amersham/Searle. Radioactivity in the effluent (0.40 ml) fractions was determined by liquid scintillation counting (Beckman LS250, Beckman Instruments). Samples for amino acid composition analysis were sealed, under vacuum in constant boiling hydrochloric acid (Pierce Chemical Co.) and hydrolyzed at 110° for 22 or 48 hr and analyses were performed on a Beckman 120 C Amino Acid analyzer.
216
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100
200
300
400
500
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, __,
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Fig. 1. Affinity chromatography of acute phase rabbit sera on a column of dimensions (1.5 × 20cm) with pneumococcal C-polysaccharide linked to Bio-Gel A-50m beads. The column was washed with 0.1 M NaCI~).2 M Tris-0.01 M CaCI2 for 24 hr prior to elution with isotonic Tris-citrate buffer at flow rate of 80 ml/hr.
s
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46o
Effluent volume, ml
Fig. 2. Chromatography of rabbit CRP (previously prepared by affÉnity chromatography) on Bio-Gel A-0.5m using Tris-buffered saline, pH 8.0. Column size 2.5 x 95 cm.
Characterization of CRP in the Rabbit RESULTS
Isolation of rabbit CRP All isolation procedures were performed at 4°C. Acute phase rabbit serum which had been clarified by filtration was passed through a CPS-agarose column (20 × 1.5 cm) which quantitatively bound the CRP. After 24 hr of washing with calcium-containing Tris-buffered saline (21.), the CRP was eluted with Tris-citrate-saline (Fig. 1). The CRP prepared in this manner was further purified by passage through Bio-Gel A-0.5m which served to remove aggregates of CRP (Fig. 2); unlike the behavior of human CRP (Osmand et al., 1975), no subunits were observed in the chromatogram.
217
equilibrated Sephadex G-25 column. The equilibration mixture contained Tris-HC1 buffer, (14C) phosphocholine and calcium chloride (0.01 M) with a total ionic strength of 0.20 as suggested by Gotschlich & Edelman (1967). Figure 4 presents Scatchard plots of binding experiments performed at 5°C and 15°C. The results of these experiments are summarized in Table 1.
Amino acid composition The results of amino acid composition studies are presented in Table 2. Relative molar ratios were calculated after suitable corrections were made for loss of amino acids during hydrolysis and are expressed as residues/subunit.
Polyacrylamide 9el electrophoresis Preparations of CRP revealed the presence of a single component at 22,900 + 400 daltons. The mol. wt of CRP was calculated by regression analysis on the basis of its behaviour during coelectrophoresis with the following standard proteins: lysozyme; fl-lactoglobulin; soy bean trypsin inhibitor; carbonic anhydrase; ovalbumin; catalase; bovine serum albumin; transferrin; and phosphorylase a (Fig. 3).
Equilibrium 9el filtration Freshly prepared CRP was applied at different concentrations in a volume of 50/~1 to the top of the
_.-Y j.,.o
DISCUSSION Rabbit CRP has been found to be readily isolated from pooled acute phase serum samples using a method of affinity chromatography on Bio-Gel beads to which C-polysaccharide has been covalently linked. Although developed for the isolation of human CRP from ascites fluid (Osmand et al., 1975), this method was highly effective for the isolation of a rabbit serum CRP in spite of the reported differences in reactivity for pneumococcal polysaccharides between human and rabbit CRP (Anderson & McCarty, 1951).
J
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Rabbit C.P
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Relative distance migrated
Fig. 3. Densitometric analysis (---) of SDS-polyacrylamide gels after coelectrophoresis of a mixture of standard proteins in the presence of CRP. Preparations of CRP revealed the presence of a single component at 22,900 + 400 daltons. The mol. wt of CRP was calculated by regression analysis on the basis of the behavior of the following standard proteins: lysozyme; /;-lactoglobulin; soy bean trypsin inhibitor; carbonic anhydrase; ovalbumin catalase; bovine serum albumin; transferrin; and phosphorylase a.
IMM. 14 3
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218
BRUCE ALLEN BACH, HENRY GEWURZ and ALEXANDER P. OSMAND
v
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Fig. 4. Scatchard plots of binding data for the interaction between rabbit CRP and (14C) phosphocholine where (v) moles of (14C) phosphocholine bound per mole of RCRP subunit (22,900 daltons) at a concentration (C) of free (14C) phosphocholine. Experiments were performed at (A) 5° and at (e) 15~C, respectively.
Freshly prepared rabbit CRP further purified by passage through Bio-Gel A-0.5m demonstrated CRP aggregates, but unlike human CRP, no free subunits were obtained. Whether this was due to a strong affinity of the subunits for agarose or the greater stability of the serum-derived rabbit CRP was not determined. The purity of CRP so prepared was established by gel filtration chromatography and SDSpolyacrylamide gel electrophoresis. The chromatographic characteristics of rabbit CRP observed in the present study were distinct from that of human CRP and are consistent with it having a mol. wt of 115,000-120,000 daltons (Somerville & Kushner, 1969), as compared with a mol. wt of 120,000-140,000 daltons for human CRP (Gotschlich & Edelman, 1965).
The migration behavior of rabbit CRP in SDSpolyacrylamide gels indicates that the molecule is composed of 5 subunits with a mol. wt of 22,900 + 400 daltons for a total mol. wt of 115,000 daltons. In this study, using rabbit CRP isolated from acute phase serum, a single component was observed. These results differ from those reported for human CRP isolated by these methods from ascites fluids which had a tool. wt of 23,300 + 500 daltons and contained a minor more rapidly migrating component (Osmand et al., 1975). Measurement of the binding affinity of rabbit CRP for (14C) phosphocholine indicated a valence of approx 1 when the subunit mol. wt of 23,000 is used. This suggests that like human CRP (Gotschlich & Edelman, 1967), rabbit CRP has one binding site per
Table 1. Binding of (14C) phosphocholine by rabbit CRP T° (°C)
K (l/mole)
5° 1.02 × 1 0 6 15° 0.62 × 1 0 6 AH° = --7.1 Kcal/mole
- AF'~ (Kcal/mole)
n (valence)
7.66 1.02 7.64 0.97 AS = 2.1 cal/deg per mol
Characterization of CRP in the Rabbit Table 2. Amino acid compositions of rabbit and human CRPs °
Asp Thr Ser Glu Pro Cys Gly Ala Val Met lie Leu Tyr Phe Lys His Arg
Rabbit
Human ~
Humanc
19.1a 8.7 19.2 19.5 10.1 n.d___l 17.6 10.3 13.4 3.8 8.4 16.7 9.2 11.7 13.1 2.9 6.1
15.2 13.0 21.8 20.2 10.4 2.0 17.0 9.2 17.1 2.0 9.5 13.8 7.8 12.6 12.3 2.0 5.8
15.8 11.1 18.9 20.9 10.1 2.0 17.0 8.2 12.1 2.0 8.2 18.1 6.7 13.2 11.9 1.9 6.7
Expressed as residues/subunit. b See Gotschlich and Edelman (1965). c See Osmand (1972). a Major differences underlined. e n.d., Not detectable. subunit. The association constants of K ° = 1.02 x 106 (1/mole) and K°s of 0.65 x 106 (1/mole) demonstrate an affinity in the intermediate range for antibody hapten reactions (Sehon, 1971), and agree well with that calculated (K -- 1.3 x 106M -1 at 5°C) for the binding of phosphocholine by human CRP (Osmand, 1972). The analysis of differences in the amino acid composition of rabbit and human CRP is of interest considering the overall similarity of these proteins. There are several significant differences in composition. The rabbit protein lacks detectable cysteine residues on amino acid analysis which is consistent with the inability to radiolabel putative cysteinyl residues upon reduction and alkylation with (14C) iodoacetamide (unpublished observation). The additional methionine residues in rabbit as compared with human CRP are consistent with a different fragmentation by CNBr of the rabbit protein as detected by gel filtration of the digested proteins (unpublished observations). Other significant differences in composition between these proteins are that rabbit CRP contains ad* Osmand A. P., Gewurz H. & Friedenson B., submitted for publication.
219
ditional aspartyl or asparaginyl residues, an additional histidine residue, while containing less threonine and valine, In this study we have utilized the structural similarity of rabbit and human CRP to extend the affinity chromatography method of pneumococcal C-polysaccharide linked to agarose for the isolation of the rabbit C-reactive protein. Equilibrium gel filtration demonstrated the strong affinity of rabbit CRP for (14C) phosphocholine and together with amino acid composition analysis provides further evidence of structural and functional similarity between C-reactive proteins generally. Additional studies in this laboratory have demonstrated extensive sequence homologies between rabbit and human C-reactive proteins.* These observations suggest that rabbit CRP provides an entirely analogous model system not only for the study of the structural characteristics of C-reactive protein, but also for its biological functions.
REFERENCES
Abernethy T. J. (1937) J. exp. Med. 65, 75. Anderson H. C. & MfCarty M. (1951) J. exp. Med. 93, 25. Eairclough G. F. Jr. & Eruton J. S. (1966) Biochemistry 5, 673. Eiedel B. A. & Gewurz H. (1976) J. lmmun. 116, 1289. Gotschlich E. C. & Edelman G. M. (1965) Proc. natn. Acad. Sci., U.S.A. 54, 558. Gotschlich E. C. & Edelman G. M. (1967) Proc. natn. Acad. Sci., U.S.A. 57, 706. Hummel J. P. & Dreyer W. J. (1962) Biochim. biophys. Acta 63, 530. Kushner I. & Somerville-Volanakis J. (1973) Proc. Soc. exp. biol. Med. 142, 112. Mortensen R. F. & Gewurz H. (1976) J. lmmun. 116, 1244. Mortensen R. F., Osmand A. P. & Gewurz H. (1975) J. exp. Med. 141, 821. Neville D. M. Jr. (1971) J. biol. Chem. 246, 6328. Osmand A. P. (1972) Ph.D. Thesis. University of Adelaide, Adelaide, So. Australia. Osmand A. P., Mortensen R. F., Siegel J. • Gewurz H. (1975) J. exp. Med. 142, 1065. Sehon A. H. In (1971) Methods in Immunology and Immunochemistry (Edited by Williams C. A. & Chase M. W.), Vol. I, p. 378. Academic Press, NY. Somerville J. & Kushner I. (1969) Biochim. biophys. Acta 207, 105. Volanakis J. E. & Kaplan M. H. (1971) Proc. Soc. exp. biol. Med. 136, 612. Weber K. & Osborn M. (1969) J. biol. Chem. 244, 4406.
C-REACTIVE PROTEIN IN THE RABBIT: ISOLATION, CHARACTERIZATION AND BINDING AFFINITY TO PHOSPHOCHOLINE* BRUCE ALLEN BACH,I" HENRY GEWURZ:~ and ALEXANDER P. OSMAND§ Department of Immunology, Rush Medical College Chicago, IL 60612, U.S.A. (Received 2 August 1976)
Abstract--Several structural and functional characteristics of rabbit C-reactive protein have been established. The protein has been isolated by affinity chromatography on pneumococcal C-polysaccharide linked to agarose. Following gel filtration, SDS-polyacrylamide gel electrophoresis indicated a subunit size of 22,900 +_ 400 daltons. Equilibrium gel filtration demonstrated a stoichiometric binding of (14C) phosphocholine with an association constant (K = 1.02 x 106M -1 at 5°C and 0.62 x 106M -~ at 15°C) in the medium range for antibody-hapten interactions. The amino acid composition for rabbit C-reactive protein is reported and compared with human C-reactive protein.
INTRODUCTION C-reactive protein (CRP) is a trace constituent of normal blood which is increased as much as 1000-fold during the acute phase of many inflammatory conditions. Additional studies in this laboratory have demonstrated the ability of CRP to fully activate the complement system (Osmand et al., 1975) and to inhibit certain lymphocyte (Mortensen et al., 1975; Mortensen & Gewurz, 1976) and platelet functions (Fiedel & Gewurz, 1976). Its unique characteristics include a calcium-dependent precipitation reaction with the pneumococcal C-polysaccharide (CPS), a cell wall teichoic acid of the pneumococcus. The specificity of the precipitation reaction of the human protein has been shown to be directed primarily towards the phosphocholine moiety (Volanakis & Kaplan, 1971). Early studies (Abernethy, 1937) in the search for experimental models for studying C-reactive protein described a C-reactive protein in the serum of monkeys with a type III pneumococcus infection. Anderson and McCarty (1951) described the presence of a C-reactive protein in rabbit serum which reacted and precipitated only with a less degraded form of C-polysaccharide, which they termed the Cx-polysaccharide. The rabbit protein was thus designated Cxreactive (CxRP). To date no further data have been presented concerning the apparent difference in reactivity between the human and rabbit proteins. Following the suggestion of Kushner and Somerville*These studies were supported by grants from the National Institutes of Health (AI 12870-02), the Leukemia Research Foundation, Inc., the Chicago Heart Association and the Hunter Trust. t Supported by a Boothroyd Medical Student Fellowship. Present address: Department of Pathology, Harvard Medical School, Boston, MA 02115. :~H.G. holds the Thomas J. Coogan, Sr. Chair in Immunology established by Marjorie Lindheimer Everett. § Address correspondence to: A. P. Osmand, Department of Immunology, Rush Medical College, Chicago, IL 60612, U.S.A. 215
Volanakis (.1973) the single term CRP will be used to refer to these proteins. This paper establishes similarities of rabbit to human CRP in terms of isolation by affinity chromatography, and binding specificity to the phosphocholine moiety of CPS as determined by equilibrium gel filtration. The extensive similarity in amino acid composition between rabbit and human CRP is also reported.
MATERIALS AND METHODS Rabbit C-reactive protein was prepared from acute phase sera essentially according to the method of Osmand et al. (1975). Sera were obtained from rabbits in the acute phase of inflammation 36-48 hr after multiple subcutaneous injections of a 1% croton oil suspension. Pooled sera were clarified by filtration through celite (Hyflo Supercel, Johns Mansville Products Corp.) and passed through an affinity column containing pneumococcal C-polysaccharide linked to Bio-Gel A-50m agarose beads (Bio Rad Laboratories). Polyacrylamide gel electrophoresis was performed exactly according to the method of Neville (1971). Gels stained with Coomassie brilliant blue according to the method of Weber & Osborn (1969) were analyzed densitometrically with a gel scanning device attached to a Beckman Acta C.V. spectrophotometer (Beckman Instruments). Binding affinities to (t4C) phosphocholine were determined by the method of Hummel & Dreyer (1962) with the modifications of Fairclough & Fruton (1966), and as redefined by Gotschlich & Edelman (1967). Binding affinity experiments were performed in jacketed columns (0.7 × 50 cm) of Sephadex (3-25 maintained at a constant temperature by a circulating water bath (Forma Scientific Co.). Radioactive (14C) phosphocholine was obtained from Amersham/Searle. Radioactivity in the effluent (0.40 ml) fractions was determined by liquid scintillation counting (Beckman LS250, Beckman Instruments). Samples for amino acid composition analysis were sealed, under vacuum in constant boiling hydrochloric acid (Pierce Chemical Co.) and hydrolyzed at 110° for 22 or 48 hr and analyses were performed on a Beckman 120 C Amino Acid analyzer.
216
BRUCE ALLEN BACH, HENRY GEWURZ and ALEXANDER P. OSMAND
If J
E c
8 "5
e0~ .o
.o
<
Washed with 0.16MNaCI
J Elu...~ 00.10MTr/~HCl with
O.OIMTril/HCl
__
pH 7J~O
.J
I
I
I
I
I
100
200
300
400
500
Effluent
, __,
4o
40O
volume, ml
Fig. 1. Affinity chromatography of acute phase rabbit sera on a column of dimensions (1.5 × 20cm) with pneumococcal C-polysaccharide linked to Bio-Gel A-50m beads. The column was washed with 0.1 M NaCI~).2 M Tris-0.01 M CaCI2 for 24 hr prior to elution with isotonic Tris-citrate buffer at flow rate of 80 ml/hr.
s
J
4(
'
46o
Effluent volume, ml
Fig. 2. Chromatography of rabbit CRP (previously prepared by affÉnity chromatography) on Bio-Gel A-0.5m using Tris-buffered saline, pH 8.0. Column size 2.5 x 95 cm.
Characterization of CRP in the Rabbit RESULTS
Isolation of rabbit CRP All isolation procedures were performed at 4°C. Acute phase rabbit serum which had been clarified by filtration was passed through a CPS-agarose column (20 × 1.5 cm) which quantitatively bound the CRP. After 24 hr of washing with calcium-containing Tris-buffered saline (21.), the CRP was eluted with Tris-citrate-saline (Fig. 1). The CRP prepared in this manner was further purified by passage through Bio-Gel A-0.5m which served to remove aggregates of CRP (Fig. 2); unlike the behavior of human CRP (Osmand et al., 1975), no subunits were observed in the chromatogram.
217
equilibrated Sephadex G-25 column. The equilibration mixture contained Tris-HC1 buffer, (14C) phosphocholine and calcium chloride (0.01 M) with a total ionic strength of 0.20 as suggested by Gotschlich & Edelman (1967). Figure 4 presents Scatchard plots of binding experiments performed at 5°C and 15°C. The results of these experiments are summarized in Table 1.
Amino acid composition The results of amino acid composition studies are presented in Table 2. Relative molar ratios were calculated after suitable corrections were made for loss of amino acids during hydrolysis and are expressed as residues/subunit.
Polyacrylamide 9el electrophoresis Preparations of CRP revealed the presence of a single component at 22,900 + 400 daltons. The mol. wt of CRP was calculated by regression analysis on the basis of its behaviour during coelectrophoresis with the following standard proteins: lysozyme; fl-lactoglobulin; soy bean trypsin inhibitor; carbonic anhydrase; ovalbumin; catalase; bovine serum albumin; transferrin; and phosphorylase a (Fig. 3).
Equilibrium 9el filtration Freshly prepared CRP was applied at different concentrations in a volume of 50/~1 to the top of the
_.-Y j.,.o
DISCUSSION Rabbit CRP has been found to be readily isolated from pooled acute phase serum samples using a method of affinity chromatography on Bio-Gel beads to which C-polysaccharide has been covalently linked. Although developed for the isolation of human CRP from ascites fluid (Osmand et al., 1975), this method was highly effective for the isolation of a rabbit serum CRP in spite of the reported differences in reactivity for pneumococcal polysaccharides between human and rabbit CRP (Anderson & McCarty, 1951).
J
/_
•
10 4
S
~
Rabbit C.P
4
E
::
1~
I
a, t
t
::
aS
S
•
' '
o
i
<
i"
,!
;
:~ :
!1
iii'
:
:
k...j: to,
I
0
I
0.5
,,
t bottom 1
Relative distance migrated
Fig. 3. Densitometric analysis (---) of SDS-polyacrylamide gels after coelectrophoresis of a mixture of standard proteins in the presence of CRP. Preparations of CRP revealed the presence of a single component at 22,900 + 400 daltons. The mol. wt of CRP was calculated by regression analysis on the basis of the behavior of the following standard proteins: lysozyme; /;-lactoglobulin; soy bean trypsin inhibitor; carbonic anhydrase; ovalbumin catalase; bovine serum albumin; transferrin; and phosphorylase a.
IMM. 14 3
t
218
BRUCE ALLEN BACH, HENRY GEWURZ and ALEXANDER P. OSMAND
v
~x
lo0
O.3
O.2
0.1
I, 0.5
1.0
V
Fig. 4. Scatchard plots of binding data for the interaction between rabbit CRP and (14C) phosphocholine where (v) moles of (14C) phosphocholine bound per mole of RCRP subunit (22,900 daltons) at a concentration (C) of free (14C) phosphocholine. Experiments were performed at (A) 5° and at (e) 15~C, respectively.
Freshly prepared rabbit CRP further purified by passage through Bio-Gel A-0.5m demonstrated CRP aggregates, but unlike human CRP, no free subunits were obtained. Whether this was due to a strong affinity of the subunits for agarose or the greater stability of the serum-derived rabbit CRP was not determined. The purity of CRP so prepared was established by gel filtration chromatography and SDSpolyacrylamide gel electrophoresis. The chromatographic characteristics of rabbit CRP observed in the present study were distinct from that of human CRP and are consistent with it having a mol. wt of 115,000-120,000 daltons (Somerville & Kushner, 1969), as compared with a mol. wt of 120,000-140,000 daltons for human CRP (Gotschlich & Edelman, 1965).
The migration behavior of rabbit CRP in SDSpolyacrylamide gels indicates that the molecule is composed of 5 subunits with a mol. wt of 22,900 + 400 daltons for a total mol. wt of 115,000 daltons. In this study, using rabbit CRP isolated from acute phase serum, a single component was observed. These results differ from those reported for human CRP isolated by these methods from ascites fluids which had a tool. wt of 23,300 + 500 daltons and contained a minor more rapidly migrating component (Osmand et al., 1975). Measurement of the binding affinity of rabbit CRP for (14C) phosphocholine indicated a valence of approx 1 when the subunit mol. wt of 23,000 is used. This suggests that like human CRP (Gotschlich & Edelman, 1967), rabbit CRP has one binding site per
Table 1. Binding of (14C) phosphocholine by rabbit CRP T° (°C)
K (l/mole)
5° 1.02 × 1 0 6 15° 0.62 × 1 0 6 AH° = --7.1 Kcal/mole
- AF'~ (Kcal/mole)
n (valence)
7.66 1.02 7.64 0.97 AS = 2.1 cal/deg per mol
Characterization of CRP in the Rabbit Table 2. Amino acid compositions of rabbit and human CRPs °
Asp Thr Ser Glu Pro Cys Gly Ala Val Met lie Leu Tyr Phe Lys His Arg
Rabbit
Human ~
Humanc
19.1a 8.7 19.2 19.5 10.1 n.d___l 17.6 10.3 13.4 3.8 8.4 16.7 9.2 11.7 13.1 2.9 6.1
15.2 13.0 21.8 20.2 10.4 2.0 17.0 9.2 17.1 2.0 9.5 13.8 7.8 12.6 12.3 2.0 5.8
15.8 11.1 18.9 20.9 10.1 2.0 17.0 8.2 12.1 2.0 8.2 18.1 6.7 13.2 11.9 1.9 6.7
Expressed as residues/subunit. b See Gotschlich and Edelman (1965). c See Osmand (1972). a Major differences underlined. e n.d., Not detectable. subunit. The association constants of K ° = 1.02 x 106 (1/mole) and K°s of 0.65 x 106 (1/mole) demonstrate an affinity in the intermediate range for antibody hapten reactions (Sehon, 1971), and agree well with that calculated (K -- 1.3 x 106M -1 at 5°C) for the binding of phosphocholine by human CRP (Osmand, 1972). The analysis of differences in the amino acid composition of rabbit and human CRP is of interest considering the overall similarity of these proteins. There are several significant differences in composition. The rabbit protein lacks detectable cysteine residues on amino acid analysis which is consistent with the inability to radiolabel putative cysteinyl residues upon reduction and alkylation with (14C) iodoacetamide (unpublished observation). The additional methionine residues in rabbit as compared with human CRP are consistent with a different fragmentation by CNBr of the rabbit protein as detected by gel filtration of the digested proteins (unpublished observations). Other significant differences in composition between these proteins are that rabbit CRP contains ad* Osmand A. P., Gewurz H. & Friedenson B., submitted for publication.
219
ditional aspartyl or asparaginyl residues, an additional histidine residue, while containing less threonine and valine, In this study we have utilized the structural similarity of rabbit and human CRP to extend the affinity chromatography method of pneumococcal C-polysaccharide linked to agarose for the isolation of the rabbit C-reactive protein. Equilibrium gel filtration demonstrated the strong affinity of rabbit CRP for (14C) phosphocholine and together with amino acid composition analysis provides further evidence of structural and functional similarity between C-reactive proteins generally. Additional studies in this laboratory have demonstrated extensive sequence homologies between rabbit and human C-reactive proteins.* These observations suggest that rabbit CRP provides an entirely analogous model system not only for the study of the structural characteristics of C-reactive protein, but also for its biological functions.
REFERENCES
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