IMMUNOLOGICAL INVESTIGATIONS, 2 1 ( 4 ) , 3 2 9 - 3 3 2 ( 1 9 9 2 )
THE EFFECT OF DIVALENT METAL IONS ON THE THERMOSTABILITY OF C3
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
S.P.Yip and W.H.P. Lewis Department of Health Sciences Hong Kong Polytechnic Hung Hom, Kowloon, Hong Kong.
ABSTRACT
Thermostability studies on human complement component C3 showed that saturation of the electronegative sites in the C3 molecule with divalent cations offered some protection against heat denaturation, a finding not previously reported in the literature. To inactivate C3 in about 30 minutes, the required temperature was 61% in the presence of excess Ca" ions, 59.572 in the presence of excess of Mg2+ions, but only 54'C in the absence of these ionized metal ions.
INTRODUCTION The complement system constitutes an integral component of host defense against infection and of the inflammatory process. The third component C3 plays a central role in the complement system since it participates in both pathways of activation and mediates many biological functions associated with the complement system (1,2). Calcium and magnesium ions are important in the complement system: Ca2' ions are required in the formation of the C1 complex, and Mg2+ions in the formation of both the classical and alternative pathway Q convertases (1). In the process of searching for
329
Copyright 0 1992 by Marcel Dekker, Inc.
YIP AND LEWIS
330
structural variation of the C3 protein using heat denaturation technique, it was found that these two cations had a profound effect on the thermostability of the C3 molecule. This article will report this interesting finding.
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
MATERIALS AND METHODS Ultrathin (0.4 mm) agarose gel was prepared using flap technique (3) in trisglycine buffer, pH 8.4 (4). The buffer contains 37 mM tris, 290 mM glycine and 1.2 mM calcium lactate. Normal serum samples were diluted 1:4 using three different solutions: tris-glycine buffer with 1.2 mM calcium lactate, pH 8.4; tris-glycine buffer with 1.2 mM magnesium sulphate, pH 8.4; and 4 mM N%EDTA, pH 7.0. The diluted samples were incubated at various temperatures for various periods of time. Note that sample dilution preceded heat treatment. The untreated and heat-treated samples (5 pL) were then
applied at the cathode side of the agarose gel and electrophoresed at 20 V/cm for 3 hours at 17'C using tris-glycine buffer, pH 8.4. At the end of the run, the gel was stained in Coomassie brilliant blue R. The effect of heat treatment was assessed qualitatively on a stained gel by comparing the intensity of the C3 band of the treated samples with that of the corresponding untreated samples.
RESULTS AND DISCUSSION Heat denaturation was initially used to test for structural variation of the C3 molecule which might be electrophoretically cryptic at alkaline pH.
No distinct
thermostability variant of C3 was revealed. However, it was found that the time required to bring about complete denaturation was very variable if heat treatment (5672) was carried out before sample dilution in the tris-glycine/calcium lactate buffer. Subsequent preliminary studies suggested that calcium ions played a crucial role in the thermostabiiity of the C3 molecule. Since magnesium ions are also involved in the activation of the complement system, it was included in the following studies. To overcome the effect of variation in the cation (Ca" and Mg2+)levels on the thermostability studies, two approaches were used: addition of excess CaZ+or Mg2+ions,
THERMOSTABILITY OF C3
TABLE 1.
331
The effects of calcium and magnesium ions on the thermostability of C3. Time (min.) taken to denature serum C3 to completion in the uresence of ~
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
Temperature ('C)
Excess Ca2'
Excess Mg2'
64
5 10
35
58
> 70
56
EDTA
>7 hr
10-15
54
25-30
53
30-35
* Not done.
or chelation of all these divalent cations in the samples. In order to make the Ca2' or Mg2' ions present in excess amount, the serum samples were diluted (1:4) in the trisglycine buffer with 1.2 mM calcium lactate or magnesium sulphate which would certainly saturate all the electronegative sites in the C3 molecule (7 pM in neat serum). On the other hand, the serum samples were diluted (1:4) in 4 mM Na,,EDTA in order to chelate all the Ca2' and Mg2' ions which are present at about 2.5 mM and 0.85 mM, respectively, in neat serum. The dilution factor and sample application volume (4 and 5 pL) were optimized for the present electrophoretic system. In the thermostability studies, diluted serum samples were incubated at different temperatures for different periods of time and then subjected to electrophoresis to compare the heat stability of the C3 protein under these conditions. The results were shown TABLE 1. It must be stressed that the effect of heat treatment was assessed qualitatively on a stained gel by comparing the intensity of the C3 band of the treated samples with that of the corresponding untreated samples. Thus, some allowances should be made for the
332
YIP AND LEWIS
visual assessment. The results showed that the C3 molecule was more heat stable when its electronegative sites were saturated with Ca" or Mg' ions. To inactivate C 3 in about 30 minutes, the required temperature was 61'C in the presence of excess Ca2' ions, 59.5'C in the presence of excess of Mg2' ions, but only 54'C in the absence of these ionized metal ions. No similar finding seems to have been reported in the
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
literature. It is speculated that interaction of the eiectronegative sites in the molecule with cations would tend to better maintain the conformation of the C3 molecule. It seems reasonable that extra energy would be required to disrupt these electrostatic interactions. Thus, for a given time, a higher temperature would be required to denature the protein. Moreover, it is also reasonable that different cations have different degrees of affinity for the electronegative sites, giving rise to different dissociation constants for the reaction. As a result, different higher temperatures would he required to denature the protein in the presence of different cations. The protective effect of these two divalent metal cations on the heat denaturation of the C3 molecule is an interesting finding, but its biological significance is not clear at the present time. Metal cations other than Ca2+or Mg2+may also have this protective effect, but these latter two are physiologically much more important and are involved in the complement system.
REFERENCES 1.
S.K.A. Law and K.B.M. Reid, Corndement. IRL Press, Oxford (1988).
2.
J.D. Lambris, Immunol. Today, 12, 387-393 (1988).
3.
B.J. Radola, Electrophoresis, 1,43-56 (1980).
4.
A. Germenis, A. Babionitakis and A. Fertakis, Vox Sang.,
a,53-55 (1982).
THE EFFECT OF DIVALENT METAL IONS ON THE THERMOSTABILITY OF C3
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
S.P.Yip and W.H.P. Lewis Department of Health Sciences Hong Kong Polytechnic Hung Hom, Kowloon, Hong Kong.
ABSTRACT
Thermostability studies on human complement component C3 showed that saturation of the electronegative sites in the C3 molecule with divalent cations offered some protection against heat denaturation, a finding not previously reported in the literature. To inactivate C3 in about 30 minutes, the required temperature was 61% in the presence of excess Ca" ions, 59.572 in the presence of excess of Mg2+ions, but only 54'C in the absence of these ionized metal ions.
INTRODUCTION The complement system constitutes an integral component of host defense against infection and of the inflammatory process. The third component C3 plays a central role in the complement system since it participates in both pathways of activation and mediates many biological functions associated with the complement system (1,2). Calcium and magnesium ions are important in the complement system: Ca2' ions are required in the formation of the C1 complex, and Mg2+ions in the formation of both the classical and alternative pathway Q convertases (1). In the process of searching for
329
Copyright 0 1992 by Marcel Dekker, Inc.
YIP AND LEWIS
330
structural variation of the C3 protein using heat denaturation technique, it was found that these two cations had a profound effect on the thermostability of the C3 molecule. This article will report this interesting finding.
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
MATERIALS AND METHODS Ultrathin (0.4 mm) agarose gel was prepared using flap technique (3) in trisglycine buffer, pH 8.4 (4). The buffer contains 37 mM tris, 290 mM glycine and 1.2 mM calcium lactate. Normal serum samples were diluted 1:4 using three different solutions: tris-glycine buffer with 1.2 mM calcium lactate, pH 8.4; tris-glycine buffer with 1.2 mM magnesium sulphate, pH 8.4; and 4 mM N%EDTA, pH 7.0. The diluted samples were incubated at various temperatures for various periods of time. Note that sample dilution preceded heat treatment. The untreated and heat-treated samples (5 pL) were then
applied at the cathode side of the agarose gel and electrophoresed at 20 V/cm for 3 hours at 17'C using tris-glycine buffer, pH 8.4. At the end of the run, the gel was stained in Coomassie brilliant blue R. The effect of heat treatment was assessed qualitatively on a stained gel by comparing the intensity of the C3 band of the treated samples with that of the corresponding untreated samples.
RESULTS AND DISCUSSION Heat denaturation was initially used to test for structural variation of the C3 molecule which might be electrophoretically cryptic at alkaline pH.
No distinct
thermostability variant of C3 was revealed. However, it was found that the time required to bring about complete denaturation was very variable if heat treatment (5672) was carried out before sample dilution in the tris-glycine/calcium lactate buffer. Subsequent preliminary studies suggested that calcium ions played a crucial role in the thermostabiiity of the C3 molecule. Since magnesium ions are also involved in the activation of the complement system, it was included in the following studies. To overcome the effect of variation in the cation (Ca" and Mg2+)levels on the thermostability studies, two approaches were used: addition of excess CaZ+or Mg2+ions,
THERMOSTABILITY OF C3
TABLE 1.
331
The effects of calcium and magnesium ions on the thermostability of C3. Time (min.) taken to denature serum C3 to completion in the uresence of ~
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
Temperature ('C)
Excess Ca2'
Excess Mg2'
64
5 10
35
58
> 70
56
EDTA
>7 hr
10-15
54
25-30
53
30-35
* Not done.
or chelation of all these divalent cations in the samples. In order to make the Ca2' or Mg2' ions present in excess amount, the serum samples were diluted (1:4) in the trisglycine buffer with 1.2 mM calcium lactate or magnesium sulphate which would certainly saturate all the electronegative sites in the C3 molecule (7 pM in neat serum). On the other hand, the serum samples were diluted (1:4) in 4 mM Na,,EDTA in order to chelate all the Ca2' and Mg2' ions which are present at about 2.5 mM and 0.85 mM, respectively, in neat serum. The dilution factor and sample application volume (4 and 5 pL) were optimized for the present electrophoretic system. In the thermostability studies, diluted serum samples were incubated at different temperatures for different periods of time and then subjected to electrophoresis to compare the heat stability of the C3 protein under these conditions. The results were shown TABLE 1. It must be stressed that the effect of heat treatment was assessed qualitatively on a stained gel by comparing the intensity of the C3 band of the treated samples with that of the corresponding untreated samples. Thus, some allowances should be made for the
332
YIP AND LEWIS
visual assessment. The results showed that the C3 molecule was more heat stable when its electronegative sites were saturated with Ca" or Mg' ions. To inactivate C 3 in about 30 minutes, the required temperature was 61'C in the presence of excess Ca2' ions, 59.5'C in the presence of excess of Mg2' ions, but only 54'C in the absence of these ionized metal ions. No similar finding seems to have been reported in the
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 01/14/15 For personal use only.
literature. It is speculated that interaction of the eiectronegative sites in the molecule with cations would tend to better maintain the conformation of the C3 molecule. It seems reasonable that extra energy would be required to disrupt these electrostatic interactions. Thus, for a given time, a higher temperature would be required to denature the protein. Moreover, it is also reasonable that different cations have different degrees of affinity for the electronegative sites, giving rise to different dissociation constants for the reaction. As a result, different higher temperatures would he required to denature the protein in the presence of different cations. The protective effect of these two divalent metal cations on the heat denaturation of the C3 molecule is an interesting finding, but its biological significance is not clear at the present time. Metal cations other than Ca2+or Mg2+may also have this protective effect, but these latter two are physiologically much more important and are involved in the complement system.
REFERENCES 1.
S.K.A. Law and K.B.M. Reid, Corndement. IRL Press, Oxford (1988).
2.
J.D. Lambris, Immunol. Today, 12, 387-393 (1988).
3.
B.J. Radola, Electrophoresis, 1,43-56 (1980).
4.
A. Germenis, A. Babionitakis and A. Fertakis, Vox Sang.,
a,53-55 (1982).
