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Polymeric binding of haemoglobin in cattle
CLINICA CHIMICA ACTA
POLYMERIC
BINDING
429
OF HAEMOGLOBIN
IN CATTLE
B. V. GOODGER
Division of Animal Health, CSIRO, Long Pocket Labovatovies, Meievs Road, Indoovoo~illy, Queensland (Australia 30 68) (Received
April 18th, 1970)
SUMMARY
Tissue damage in cattle appears to induce the production of an abnormal serum protein whose function is to bind haemoglobin. Both the abnormal protein and the protein-haemoglobin complex exist in polymerized forms similar to human haptoglobin
2-2.
INTRODUCTION
It is thought that the haptoglobin gene (Hp2) which controls the polymeric form of haptoglobin (HP), occurs solely in human being+. In all other mammals studied so far, which included the great apes, Hp appears to exist only in the monomer form, similar to human Hp I-I (ref. I)., In cattle, Bremne? showed that Hp existed in serum at an extremely low concentration and that it was difficult to demonstrate. He injected several calves with turpentine and was then able to demonstrate the haptoglobin-haemoglobin (Hp-Hb) complex in serum using paper electrophoresis to separate the complex from free Hb. In the course of a more generalized study of the nature of a high molecular weight complex that was observed in the serum of cattle after erythrocyte damage3, it was necessary to characterize the bovine Hp-Hb complex. The results of these observations are given in this paper. MATERIALS
AND METHODS
Pre$aration of ha$toglobin-ewiched semwn To prepare HP-enriched serum, calves weighing 200-250 lbs in weight were each given one subcutaneous injection of 4.5 ml of oil of turpentine. Serum samples were collected I day before and 3 days after the injection. Gel jiltratios chromatogra@y Gel filtration of bovine
serum or serum fractions
was performed
on a
15”
x I”
Clin. Chim. Acta, zg (1970) qzg-435
430
GOODGER
column of Sephadex G-zoo superfine. Columns were eluted with 0.05 M Tris-HCl buffer pH 8.0, at a flow rate of 5 ml/h. Eluate was collected in 5-ml fractions. The absorbance (A) of each fraction was read at 2800 A and 4130 A.. Electrophoresis Disc electrophoresis
was performed on Shandon Disc Electrophoresis Apparatus *, according to the method of Davis4. A modification was that a linear gradient from 3.5 to 7% acrylamide was used in the running gel. This gradient was prepared with a Buchler Gradient Mixer. Acrylamide electro$horesis was performedusing a Gradipore apparatus* *. A slab of 4-26% concave gradient acrylamide gel was used for each electrophoretic run. Staining
of electrophoretic
patterns
Electrophoretic patterns were stained for protein with 1% in 7% acetic acid5 and for haemoglobin with benzidine-hydrogen Density
Amido Black IOB peroxides.
gradient centrifugation 0.15 ml of samples were subjected to ultracentrifugation on 1o--37~/~ linear
0
4 Effluent
8
12
in 5-ml
16
20
iractions
Fig. I. Gel filtration on Sephadex G-zoo of a crude Hp-Hb complex obtained by ammonium after injection with turpentine, and sulphate precipitation of a mixture of serum, obtained homologous oxy-Hb. One ml of the crude complex was applied to a 15” x I” column of Sephadex G-zoo. Eluting buffer was 0.05 M Tris-HCl pH 8.0, flow rate was 5 ml/h, and the eluate was collected in 5-ml fractions. The A of each fraction was taken at 2800 A and 4130 A. Fig. z. Disc acrylamide electrophoresis of a partly purified Hp-Hb complex obtained by gel filtration. A linear acrylamide gradient of 3.5% to 7% was used and separated fractions were stained with benzidine-hydrogen peroxide. * Shandon **
Townson
czin.
Scientific & Mercer
Chinz. Acta,
Co. Ltd.,
London.
(Distributors)
29 (1970)
429-435
Pty.
Ltd.,
Sydney,
New
South Wales,
Australia.
BINDING
OF
Hb
431
IN CATTLE
sucrose gradients using a Beckman SW3gL rotor at 34000 rev./min for 18 h. After centrifugation, the bottoms of the tubes were pierced and fractions of 20 drops each were collected with the aid of a Buchler Density Gradient Fraction Collector. RESULTS
Homologous oxy-Hb was added to serum taken from an animal, 3 days after a subcutaneous injection with turpentine. That part of the serum-Hb mixture which precipitated between 5070% saturation with ammonium sulphate was then subjected to gel filtration on Sephadex G-200 using 0.05 M tris-HCl pH 8.0 as eluting buffer. The Hp-Hb complex was eluted as a homogeneous fraction at the void volume (Fig. I), being completely free of unbound Hb which was retarded on the Sephadex column. Cellulose acetate electrophoresis of the partly purified Hp-Hb complex showed it migrated as a homogeneous band with a mobility between those of BZ- and PIglobulins. Disc acrylamide electrophoresis in a 3.5% to 7.0% linear acrylamide gradient revealed the molecular heterogeneity of the Hp-Hb complex as at least 8 protein bands were resolved (Fig. 2) each of which could be selectively stained for Hb with benzidine-hydrogen peroxide. Further evidence of heterogeneity in size was obtained by density gradient centrifugation. Centrifugation of the Hp-Hb complex in a 10-37O/~ linear sucrose gradient using a Beckman SW39L rotor at 34000 rev./min for 18 h gave a series of pigmented bands, of which 5 were easily visible, extending from the centre to the bottom of the gradient (Fig. 3). The gradients were collected in zo-drop fractions
Fig. 3. Patterns in a 1~37% linear sucrose gradient of (a) serum from a calf after injection turpentine, (b) partly purified Hp-Hb complex, (c) homologous oxy-Hb.
with
by piercing the bottom of the cellulose nitrate tube. The gradient fractions were then electrophoresed in slab acrylamide using a 4-24% concave acrylamide gradient. Subsequent staining with benzidine-hydrogen peroxide (Fig. 4) showed the relation of the density gradient fractions with respect to electrophoretic mobility in acrylamide. To partly characterize the Hp component, a sample of HP-rich serum was fractionated on Sephadex G-200 (Fig. 5). Homologous oxy-Hb was then added to Cl&. Chim. Acta, zg (1970)
429-435
GOODGER
432
Fig. 4. Slab acrylamide electrophoresis of fractions obtained from density gradient centrifugation of the Hp-Hb complex. Twenty fractions were collected, starting at the bottom of the gradient. After electrophoresis, the separated fractions were stained with benzidine-hydrogen peroxide.
each Sephadex fraction and the resulting mixtures were electrophoresed on slab acrylamide and stained with benzidine-hydrogen peroxide. The first three Sephadex fractions were the only ones in which the polymers of the Hp-Hb complex were demonstrated. Fig. 6 shows the electrophoretic patterns of the first three Sephadex fractions stained (A) with benzidine-hydrogen peroxide and (B) with Amido Black IOB. From these results it appears that the Hp exists in a polymeric form prior to
2.0.
1.5.
4 :: w 7 1.0. I
0.5 -
0
L It
8
12
-3
in 5-ml frcxtic
L ,“S
Fig. 5. Gel filtration on a 15” x I” Sephadex G-zoo column of I ml of serum taken from an animal 3 days after injection with turpentine. The shaded portion shows those fractions in which Hp was detected. Clin. Chim. Acta,
29 (1970)
429-435
BINDING OF
Hb IN CATTLE
433
Fig. 6. (A) Slab acrylamide electrophoresis of Sephadex fractions I, z and 3 obtained as shown in Fig. 5. The fractions to the left of the centre had oxy-Hb added prior to electrophoresis, Staining was with benzidin~hydrogen peroxide. (B) As fox (A) but counterstaine~ with Amiclo Black IOB.
Hb attachment and that the base polymer must have a molecular weight of at least 200 000. Homologous oxy-Hb was also added to the fractions obtained from density gradient centrifugation of a sample of HP-rich serum. Resultant slab acrylamide electrophoresis and subsequent staining with benkline-hydrogen peroxide as seen in Fig. 7 indicates the unbound Hp is tocated in the region of ~~-30% sucrose and appears to exist in polymeric forms. Sera from 50 normal calves were examined for the polymeric HP-binding protein. Oxy-Hb was added to each serum which was then analysed by disc electrophoresis and benzidine-hydrogen peroxide staining. In none of the sera could the polymeric protein be detected. Cl&z.. Chim.
Acta,
zg (rgyo)
@g-+35
GOODGER
Fig. 7. Slab acrylamide electrophoresis and benzidine-hydrogen peroxide gradient fractions of serum obtained after injection with turpentine.
staining
of density
DISCUSSION
From these results it appears that the cow possesses a Hb-binding protein similar to human Hp z-z. Although homogeneous as regards electrical charge, it consists of a series of polymers which may be demonstrated by ultracentrifugation and molecular sieve electrophoresis. However, the protein in cattle, analogous to Hp 2-2, may not be a normal plasma protein and may be produced only in response to tissue damage. In sera from 50 normal calves to which homologous Hb had been added, no evidence of Hb-binding in a polymeric form could be demonstrated. From this it must be assumed that normal bovine serum either does not contain the polymeric Hb-binding protein or that the concentration of the protein is too low to permit detection on acrylamide gel by available methods. Neuhaus and Sogoian7 identified Hp in serum of normal cattle by electrophoretic differences between the Hp-Hb complex and free Hb in agar gel electrophoresis. They estimated a molecular weight of 85000 which is of the same order or size as the Hp I-I type. As the base polymer of the Hb-binding protein reported here is at least 200000 as estimated by gel filtration (Fig. 5), it cannot be the same component identified by the above workers. Tissue damage has been invoked in only four animals and identical polymerized forms of bound Hb were found in the serum of the four animals after addition of Hb. Similar polymeric forms were not found in the serum prior to tissue damage. Even though the sera of only a smallnumber of animals has been tested, the finding of the polymeric Hb-binding protein in each sera would not be expected if the protein were a genetic variant of normal Hp. Therefore, this protein, similar to human Hp 2-2, may not be a genetic variant but a serum constituent produced by all cattle in response to stress, the function of which is to increase the low Hb-binding capacity of normal bovine serum. ACKNOWLEDGMENTS
I wish to thank Dr. D. F. Mahoney, Principal Research Scientist, CSIRO, Clin.
Chim
A&,
zg (1970)
qzg-435
BINDING
OF
Hb IN
435
CATTLE
Long Pocket Laboratories, for his helpful advice and criticism during the course of this work; Mr. G. B. Mirre and Mr. K. Rode-Bramanis for able technical assistance; Mr. K. Rode-Bramanis for relevant photography. The work was supported in part by funds from the Australian Meat Research Committee. REFERENCES I H. E. SCHULTZE AND J. F. HEREMANS, in Molecular Biology of Human Proteins, Vol. I, Amsterdam, 1966, p. 389. 2 K. C. BREMNER. Australian I. Exfitl. Bid. Med. Sk., 42 (1964) 641. 3 D. F. MAHONEY AND B. V. (.?OOD&ER, Exptl. Parasitol.; 24 jI$g) ‘;75. 4 B. J. DAVIS, Ann. N.Y. Acad. Sci., 121 (1964) 404. 5 R. J. WIEME, in Agar Gel Electrofihoresis, Elsevier, Amsterdam, 1965, p. 140. 6 R. J. WIEME, in Agar Gel Electro~horesis, Elsevier, Amsterdam, 1965. p. 153. 7 0. W. NEUHAUS AND V. P. SOGOIAN, Nature, 192 (1961) 558.
Elsevier,
Clin. Chim Acta, 2g (1970) 429-435
POLYMERIC
BINDING
429
OF HAEMOGLOBIN
IN CATTLE
B. V. GOODGER
Division of Animal Health, CSIRO, Long Pocket Labovatovies, Meievs Road, Indoovoo~illy, Queensland (Australia 30 68) (Received
April 18th, 1970)
SUMMARY
Tissue damage in cattle appears to induce the production of an abnormal serum protein whose function is to bind haemoglobin. Both the abnormal protein and the protein-haemoglobin complex exist in polymerized forms similar to human haptoglobin
2-2.
INTRODUCTION
It is thought that the haptoglobin gene (Hp2) which controls the polymeric form of haptoglobin (HP), occurs solely in human being+. In all other mammals studied so far, which included the great apes, Hp appears to exist only in the monomer form, similar to human Hp I-I (ref. I)., In cattle, Bremne? showed that Hp existed in serum at an extremely low concentration and that it was difficult to demonstrate. He injected several calves with turpentine and was then able to demonstrate the haptoglobin-haemoglobin (Hp-Hb) complex in serum using paper electrophoresis to separate the complex from free Hb. In the course of a more generalized study of the nature of a high molecular weight complex that was observed in the serum of cattle after erythrocyte damage3, it was necessary to characterize the bovine Hp-Hb complex. The results of these observations are given in this paper. MATERIALS
AND METHODS
Pre$aration of ha$toglobin-ewiched semwn To prepare HP-enriched serum, calves weighing 200-250 lbs in weight were each given one subcutaneous injection of 4.5 ml of oil of turpentine. Serum samples were collected I day before and 3 days after the injection. Gel jiltratios chromatogra@y Gel filtration of bovine
serum or serum fractions
was performed
on a
15”
x I”
Clin. Chim. Acta, zg (1970) qzg-435
430
GOODGER
column of Sephadex G-zoo superfine. Columns were eluted with 0.05 M Tris-HCl buffer pH 8.0, at a flow rate of 5 ml/h. Eluate was collected in 5-ml fractions. The absorbance (A) of each fraction was read at 2800 A and 4130 A.. Electrophoresis Disc electrophoresis
was performed on Shandon Disc Electrophoresis Apparatus *, according to the method of Davis4. A modification was that a linear gradient from 3.5 to 7% acrylamide was used in the running gel. This gradient was prepared with a Buchler Gradient Mixer. Acrylamide electro$horesis was performedusing a Gradipore apparatus* *. A slab of 4-26% concave gradient acrylamide gel was used for each electrophoretic run. Staining
of electrophoretic
patterns
Electrophoretic patterns were stained for protein with 1% in 7% acetic acid5 and for haemoglobin with benzidine-hydrogen Density
Amido Black IOB peroxides.
gradient centrifugation 0.15 ml of samples were subjected to ultracentrifugation on 1o--37~/~ linear
0
4 Effluent
8
12
in 5-ml
16
20
iractions
Fig. I. Gel filtration on Sephadex G-zoo of a crude Hp-Hb complex obtained by ammonium after injection with turpentine, and sulphate precipitation of a mixture of serum, obtained homologous oxy-Hb. One ml of the crude complex was applied to a 15” x I” column of Sephadex G-zoo. Eluting buffer was 0.05 M Tris-HCl pH 8.0, flow rate was 5 ml/h, and the eluate was collected in 5-ml fractions. The A of each fraction was taken at 2800 A and 4130 A. Fig. z. Disc acrylamide electrophoresis of a partly purified Hp-Hb complex obtained by gel filtration. A linear acrylamide gradient of 3.5% to 7% was used and separated fractions were stained with benzidine-hydrogen peroxide. * Shandon **
Townson
czin.
Scientific & Mercer
Chinz. Acta,
Co. Ltd.,
London.
(Distributors)
29 (1970)
429-435
Pty.
Ltd.,
Sydney,
New
South Wales,
Australia.
BINDING
OF
Hb
431
IN CATTLE
sucrose gradients using a Beckman SW3gL rotor at 34000 rev./min for 18 h. After centrifugation, the bottoms of the tubes were pierced and fractions of 20 drops each were collected with the aid of a Buchler Density Gradient Fraction Collector. RESULTS
Homologous oxy-Hb was added to serum taken from an animal, 3 days after a subcutaneous injection with turpentine. That part of the serum-Hb mixture which precipitated between 5070% saturation with ammonium sulphate was then subjected to gel filtration on Sephadex G-200 using 0.05 M tris-HCl pH 8.0 as eluting buffer. The Hp-Hb complex was eluted as a homogeneous fraction at the void volume (Fig. I), being completely free of unbound Hb which was retarded on the Sephadex column. Cellulose acetate electrophoresis of the partly purified Hp-Hb complex showed it migrated as a homogeneous band with a mobility between those of BZ- and PIglobulins. Disc acrylamide electrophoresis in a 3.5% to 7.0% linear acrylamide gradient revealed the molecular heterogeneity of the Hp-Hb complex as at least 8 protein bands were resolved (Fig. 2) each of which could be selectively stained for Hb with benzidine-hydrogen peroxide. Further evidence of heterogeneity in size was obtained by density gradient centrifugation. Centrifugation of the Hp-Hb complex in a 10-37O/~ linear sucrose gradient using a Beckman SW39L rotor at 34000 rev./min for 18 h gave a series of pigmented bands, of which 5 were easily visible, extending from the centre to the bottom of the gradient (Fig. 3). The gradients were collected in zo-drop fractions
Fig. 3. Patterns in a 1~37% linear sucrose gradient of (a) serum from a calf after injection turpentine, (b) partly purified Hp-Hb complex, (c) homologous oxy-Hb.
with
by piercing the bottom of the cellulose nitrate tube. The gradient fractions were then electrophoresed in slab acrylamide using a 4-24% concave acrylamide gradient. Subsequent staining with benzidine-hydrogen peroxide (Fig. 4) showed the relation of the density gradient fractions with respect to electrophoretic mobility in acrylamide. To partly characterize the Hp component, a sample of HP-rich serum was fractionated on Sephadex G-200 (Fig. 5). Homologous oxy-Hb was then added to Cl&. Chim. Acta, zg (1970)
429-435
GOODGER
432
Fig. 4. Slab acrylamide electrophoresis of fractions obtained from density gradient centrifugation of the Hp-Hb complex. Twenty fractions were collected, starting at the bottom of the gradient. After electrophoresis, the separated fractions were stained with benzidine-hydrogen peroxide.
each Sephadex fraction and the resulting mixtures were electrophoresed on slab acrylamide and stained with benzidine-hydrogen peroxide. The first three Sephadex fractions were the only ones in which the polymers of the Hp-Hb complex were demonstrated. Fig. 6 shows the electrophoretic patterns of the first three Sephadex fractions stained (A) with benzidine-hydrogen peroxide and (B) with Amido Black IOB. From these results it appears that the Hp exists in a polymeric form prior to
2.0.
1.5.
4 :: w 7 1.0. I
0.5 -
0
L It
8
12
-3
in 5-ml frcxtic
L ,“S
Fig. 5. Gel filtration on a 15” x I” Sephadex G-zoo column of I ml of serum taken from an animal 3 days after injection with turpentine. The shaded portion shows those fractions in which Hp was detected. Clin. Chim. Acta,
29 (1970)
429-435
BINDING OF
Hb IN CATTLE
433
Fig. 6. (A) Slab acrylamide electrophoresis of Sephadex fractions I, z and 3 obtained as shown in Fig. 5. The fractions to the left of the centre had oxy-Hb added prior to electrophoresis, Staining was with benzidin~hydrogen peroxide. (B) As fox (A) but counterstaine~ with Amiclo Black IOB.
Hb attachment and that the base polymer must have a molecular weight of at least 200 000. Homologous oxy-Hb was also added to the fractions obtained from density gradient centrifugation of a sample of HP-rich serum. Resultant slab acrylamide electrophoresis and subsequent staining with benkline-hydrogen peroxide as seen in Fig. 7 indicates the unbound Hp is tocated in the region of ~~-30% sucrose and appears to exist in polymeric forms. Sera from 50 normal calves were examined for the polymeric HP-binding protein. Oxy-Hb was added to each serum which was then analysed by disc electrophoresis and benzidine-hydrogen peroxide staining. In none of the sera could the polymeric protein be detected. Cl&z.. Chim.
Acta,
zg (rgyo)
@g-+35
GOODGER
Fig. 7. Slab acrylamide electrophoresis and benzidine-hydrogen peroxide gradient fractions of serum obtained after injection with turpentine.
staining
of density
DISCUSSION
From these results it appears that the cow possesses a Hb-binding protein similar to human Hp z-z. Although homogeneous as regards electrical charge, it consists of a series of polymers which may be demonstrated by ultracentrifugation and molecular sieve electrophoresis. However, the protein in cattle, analogous to Hp 2-2, may not be a normal plasma protein and may be produced only in response to tissue damage. In sera from 50 normal calves to which homologous Hb had been added, no evidence of Hb-binding in a polymeric form could be demonstrated. From this it must be assumed that normal bovine serum either does not contain the polymeric Hb-binding protein or that the concentration of the protein is too low to permit detection on acrylamide gel by available methods. Neuhaus and Sogoian7 identified Hp in serum of normal cattle by electrophoretic differences between the Hp-Hb complex and free Hb in agar gel electrophoresis. They estimated a molecular weight of 85000 which is of the same order or size as the Hp I-I type. As the base polymer of the Hb-binding protein reported here is at least 200000 as estimated by gel filtration (Fig. 5), it cannot be the same component identified by the above workers. Tissue damage has been invoked in only four animals and identical polymerized forms of bound Hb were found in the serum of the four animals after addition of Hb. Similar polymeric forms were not found in the serum prior to tissue damage. Even though the sera of only a smallnumber of animals has been tested, the finding of the polymeric Hb-binding protein in each sera would not be expected if the protein were a genetic variant of normal Hp. Therefore, this protein, similar to human Hp 2-2, may not be a genetic variant but a serum constituent produced by all cattle in response to stress, the function of which is to increase the low Hb-binding capacity of normal bovine serum. ACKNOWLEDGMENTS
I wish to thank Dr. D. F. Mahoney, Principal Research Scientist, CSIRO, Clin.
Chim
A&,
zg (1970)
qzg-435
BINDING
OF
Hb IN
435
CATTLE
Long Pocket Laboratories, for his helpful advice and criticism during the course of this work; Mr. G. B. Mirre and Mr. K. Rode-Bramanis for able technical assistance; Mr. K. Rode-Bramanis for relevant photography. The work was supported in part by funds from the Australian Meat Research Committee. REFERENCES I H. E. SCHULTZE AND J. F. HEREMANS, in Molecular Biology of Human Proteins, Vol. I, Amsterdam, 1966, p. 389. 2 K. C. BREMNER. Australian I. Exfitl. Bid. Med. Sk., 42 (1964) 641. 3 D. F. MAHONEY AND B. V. (.?OOD&ER, Exptl. Parasitol.; 24 jI$g) ‘;75. 4 B. J. DAVIS, Ann. N.Y. Acad. Sci., 121 (1964) 404. 5 R. J. WIEME, in Agar Gel Electrofihoresis, Elsevier, Amsterdam, 1965, p. 140. 6 R. J. WIEME, in Agar Gel Electro~horesis, Elsevier, Amsterdam, 1965. p. 153. 7 0. W. NEUHAUS AND V. P. SOGOIAN, Nature, 192 (1961) 558.
Elsevier,
Clin. Chim Acta, 2g (1970) 429-435