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Binding of copper to albumin and participation of cysteine in vivo and in vitro
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 2’73, No. 2, September, pp. 572-5’77,1989
Binding of Copper to Albumin and Participation of Cysteine in Vivo and in Vitro KAZUO T. SUZUKI,**1 AKIRA
KARASAWA,**t
AND
KEI YAMANAKAT
*National Institute for Environmental Studies, 0nogawa, Tsukuba, I&-ski 305, Japan, and ~Institute of Environmental Sciences, University of Tsukuba, Amakubo, Tsukuba 305, Japan Received April 7,1989, and in revised form May 22,1989
Albumin, the major copper-binding protein in blood serum, was shown to form different albumin-copper complexes in in vivo and in vitro. Cupric ions added in vitro to control rat serum bound preferentially to mercaptalbumin and the mercaptalbumin-copper complex remained unchanged with time. Cupric ions injected intravenously into the rat first formed the mercaptalbumin-copper complex; this binary complex changed gradually with time to form an albumin-copper-cysteine complex. The participation of cysteine in the formation of this complex was demonstrated in vitro and further suggested that its conversion was an oxidative reaction. Glutathione also participated in forming the complex, but it was not as effective as cysteine. Albumin-copper complexes were separated on a gel filtration column and detected simultaneously by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry. 0 1989 Academic Press. Inc.
Albumin is a major binding protein for metal ions in blood serum and several binding sites have already been characterized for some metals (l-3). Copper is one of the metals that bind to albumin in the blood. Over 90% of the Cu in human plasma is firmly bound to an az-globulin, ceruloplasmin, and the remainder is less firmly bound in large part to albumin (4, 5). However, this does not mean that ceruloplasmin is the major carrier protein for the transport of Cu absorbed in the blood plasma (6). Copper in ceruloplasmin is incorporated during its synthesis in the liver (7) and Cu bound to ceruloplasmin does not exchange or equilibrate with ionic Cu in vitro (8). Therefore, although Cu bound to albumin is less than 10% of plasma Cu, albumin is probably the largest labile pool for plasma Cu and may be the most important Cu carrier component in blood serum (4). The Cu(II)-transport site of serum albumin has been extensively studied and has
1 To whom correspondence
should be addressed.
0003-9861/89 $3.00 Copyright All rights
0 1989 hy Academic Press, Inc. of reproduction in any form resewed.
been suggested to involve the a-amino nitrogen, two intervening peptide nitrogens, and the imidazole nitrogen of the histidine residue in position 3 (9-13). Extensive investigations in the Foster’s laboratory contributed to clarify the structure and conformation of plasma albumin as compiled in “Selected Papers on Plasma Albumin” by his students (14). Plasma albumin is a mixture of mercaptalbumin and nonmercaptalbumin, the two forms of human albumin have been separated on a high-performance liquid chromatograph using an Asahipak GS-520 column (15). Mercaptalbumin and nonmercaptalbumin in rat plasma have been separated on the same column under different conditions and mercaptalbumin was identified as the serum carrier for Cd (16). Metals bound firmly to specific binding sites of metalloproteins can be separated without dissociation from the proteins during separation procedures. However, metals bound less firmly to proteins may interact with ligands of column resins, resulting in redistribution or dissociation of 572
COPPER-ALBUMIN-CYSTEINE
COMPLEX IN RAT SERUM
573
metal ions. Silanol groups in silica gels when 12 weeks old (body weight, approximately 290 g). Cupric chloride in physiological saline was inmay work as efficient anionic counterparts of metallic cations and gel filtration col- jected into the femoral vein (three to five rats per umns without silica gels are preferable for group) as a singledoseof 0.8 mgCu/kg body wt under anesthesia. The rats were killed 5, 10, the separation of metal-binding proteins. pentobarbital 60, and 180 min later by exsanguination. The blood An Asahipak GS-520 column used for sep- was centrifuged at 23OOgfor 10 min and the serum aration of mercaptalbumin and nonmer- was analyzed immediately by the HPLC-ICP method captalbumin is made of resins without sil- (16-18). Control serum was prepared from rats withica gels and can be effectively used to sepa- out any treatment. rate metal-binding proteins (16). In vitro treatments. A series of experiments were Biological samples contain a variety of carried out using fresh control serum each time. Cupric chloride and/or cysteine were added to the serum elements and these elements interact with to concentrations of 8 pg Cu/ml serum and/or 80 pg each other in the body. Hence, the simultacysteine/ml serum. The serum was incubated at 37°C neous detection of multi-elements is someaerobically without sealing the test tube or anaerobitimes essential to understand biological cally with sealing the test tube in an atmosphere of reactions in which metal ions are involved. nitrogen gas. We have developed an efficient analytical Analysis bg the HPLC-ICP method (16-18). A &l-ml method for the simultaneous determinaportion of serum was applied to an Asahipak GS-520 tion of elements in biological samples; an column (7.6 X 500 mm, Asahi Chemical Co. Ltd., Toinductively coupled argon plasma-atomic kyo) and the column was eluted with 10 mM Tris/HCl emission spectrometer has been used buffer solution (pH 7.4, containing 0.9% NaCl and as a multi-element-specific detector with 0.05% NaNa, the dissolved gases were removed before use with a degasser) at a flow rate of 1.0 ml/min on HPLC (16-18). During analysis of Cu in blood serum, we an HPLC (Model 340, Beckman). Concentrations of multi-elements in the eluate were determined simulobserved that Cu-albumin complexes taneously using an ICP spectrometer (JY48 PVH, formed in viva and in vitro show different Seiko Instruments & Electronics, Ltd., Tokyo) as a decharacteristics on a gel filtration column tector of HPLC. Absorbances at 254 and 280 nm were by the above HPLC-ICP’ method. Cupric monitored with a dual-wavelength uv detector (Model ions added in vitro to rat serum were bound 152, Altex, Berkeley). The distribution profiles were preferentially to mercaptalbumin and the drawn using a personal computer (PC-9801, NEC Cu-mercaptalbumin complex remained Corp., Tokyo) and an XY-plotter (FP5301R, Graphtec unchanged with time. On the other hand, Corp., Tokyo). the Cu-albumin complex formed in vivo changed with time (19). This difference beRESULTS tween in viva and in vitro reactions was obUnder the present column conditions, served only when mercaptalbumin and nonmercaptalbumin were separated, and serum proteins were largely separated into when Cu and albumin forms were sepa- globulins (eluted before 15.0 min) and alrated as metal-binding complexes. The bumins (eluted after 15.6 min). Sulfur was metal and proteins have to be detected si- detected with an ultravacuum ICP specmultaneously for this purpose. The present trometer as shown in Fig. 1B. The distribustudy was undertaken to clarify the con- tion of sulfur can be used as an efficient proteins tributing factors in the formation of tool to detect sulfur-containing different Cu-albumin complexes between and low-molecular-weight compounds; the in viva and in vitro systems utilizing the highest sulfur peak at 15.2 min in the control profile in Fig. 1 was assigned to merHPLC-ICP method. captalbumin, while a broad shoulder peak MATERIALS AND METHODS at around 19.0 min was assigned to nonmercaptalbumin (16). The shoulder peak at Animals Female rats of the Wiatar strain were 17.0 min and a peak at 18.1 min in the conpurchased from Clea Japan Co., Tokyo, and were used trol sulfur profile were due to sulfate ions and glutathione, respectively (checked by using appropriate standards). The sulfur x Abbreviation used: ICP, inductiveiy coupled argon distribution can be used as a comparable plasma-atomic emission spectrometry,
574
SUZUKI,
KARASAWA,
B 5 pg/ml
A n I
I....I....I....I
10
..l....l....‘....
15
Retention
20 time
25 (mid
10 Retention
15
20 time
(mid
FIG. 1. Changes in Cu and sulfur distributions in rat serum after a single intravenous injection of cupric chloride. A and B show the distribution profiles of Cu and sulfur, respectively, in rat serum. The animals were injected intravenously with CuCla at a dose of 0.8 mg Cu/kg and were killed 5 and 10 min and 1 and 3 h later. The vertical bars indicate the detection level of Cu or sulfur by the HPLC-ICP method. Peaks are assigned as follows: ceruloplasmin (12.7 min in A), mercaptalbumin (15.2 min in A and B), sulfate ion (17.0 min in B), glutathione (18.1 min in B), nonmercaptalbumin (19.0 min in A and B).
or superior tool to the absorbances at 254 and 280 nm (Fig. 2B). Copper in rat serum was bound mostly to ceruloplasmin (retention time 12.7 min) and only in a small amount to albumin (15.3 min) as shown in Fig. 1. However, cupric ions injected intravenously into the rat did not distribute to ceruloplasmin and was bound mostly to albumin. Copper distributed to albumin was eluted separately into two peaks; one was eluted at 15.2 min (at the position of mercaptalbumin) and the other at 17.0 min (at the position between mercaptalbumin and nonmercaptalbumin) (Fig. lA, 5 min). The relative intensities of these two Cu peaks changed with time, while the intensity of ceruloplasmin remained constant. In accordance with the change in Cu distribution in the albumin fraction., sulfur distribution in the albumin fraction also changed dramatically. However, the sulfur distribution in the globulin fraction was not altered (Fig. 1B). When the sulfur distribution in the albumin fraction was examined precisely, the change in sulfur distribution can be summarized as follows: mercaptalbumin was converted rapidly with time to a different form of al-
AND
YAMANAKA
bumin in the presence of Cu and the altered form of albumin started to return to the original mercaptalbumin when Cu disappeared from the blood serum. Although the distributions of Cu and sulfur and absorbances at 254 and 280 nm (data not shown) in the albumin fraction changed with time after injection of cupric ion into the rat, these changes were not observed when cupric ions were added in witro into the control serum as shown in Fig. 2. Copper was bound to mercaptalbumin and the albumin peaks as monitored by the distribution profiles of sulfur and absorbances at 254 and 280 nm remained unchanged. The mercaptalbumin-Cu complex first formed in viva was assumed to be converted into an altered form of albumin-Cu complex. The altered form of albuminCu complex was eluted slower than the mercaptalbumin-Cu complex and faster than the nonmercaptalbumin-Cu complex, which suggested the participation of a hydrophilic low-molecular-weight compound in the rat serum in this conversion. Cysteine was examined as a candidate comnound in the blood serum, as shown in Fig. 3.
g-fJe 382 1 Omin
30min
FIG. 2. Changes in Cu and sulfur distributions in rat serum after an in vitro addition of cupric chloride. Cupric chloride was added to the control serum to an amount of 8 Irg Cu/ml serum and then incubated at 37°C for 10 or 30 min. A shows the distribution profiles of Cu, while the upper two profiles and lower three profiles in B show the distribution profiles of absorbances at 254 and 280 nm, and sulfur for the control, respectively. The vertical bars indicate the detection level of Cu or sulfur. Peaks are assigned as indicated in the legend to Fig. 1.
COPPER-ALBUMIN-CYSTEINE
IN RAT
lJ$&
AL
Omin
min
Omin
1Omin
Lmin
lJdL
1 Omin
Retention
25
time (mid
RZentiZ-4
tin?: (mi3
FIG. 3. Changes in Cu distribution in rat serum after an in vitro addition of cupric chloride and cysteine. Cupric chloride and cysteine were added in vitro to the control serum to amounts of 8 pg Cu/ml serum and 80 pg/ml serum, respectively, and then the serum solutions were incubated at 37°C for 5,10,20, and 30 min aerobically (A) or anaerobically (B; bubbled through with 99.999% nitrogen gas and then sealed in an atmosphere of nitrogen gas). Peaks are assigned as indicated in the legend to Fig. 1.
I....,,..,,,,
15
Retention
II
20
Smin
1OWl
i/-L
I....,....,..,
30min 15
skb& Omin
30min
10 20min
10
I 5 pa/ml
B
Onun
3%k &
A
575
SERUM
5 fig/ml
I. 05 ye/ml
CU
COMPLEX
20
time
(mid
10
15
Retention
20
time
(min)
FIG. 4. Changes in sulfur distribution in rat serum after an in vitro addition of cupric chloride and cysteine. See the legend to Fig. 3. The vertical bars indicate the detector level of sulfur. Peaks are assigned as indicated in the legend to Fig. 1.
lar-weight compound in the serum, as shown in Figs. 5 and 6. Similar changes in the distributions of Cu and sulfur were also observed by the addition of glutathi-
Cupric ions and cysteine were added in vitro into the control serum and distribu-
tions of Cu and sulfur were determined by the HPLC-ICP method after incubation for 5, 10, 20, and 30 min. Although dissolved oxygen gas might not be removed completely, the incubation was also carried out in an atmosphere of nitrogen gas after bubbling through with nitrogen gas. Copper added in vitro without cysteine remained unchanged as mercaptalbumin-Cu complex even after 30 min (Fig. 2). However, an addition of cysteine into the control serum along with cupric ions induced changes in the distributions of both Cu and sulfur as shown in Figs. 3 and 4. The intensities of Cu and sulfur peaks of the mercaptalbumin-Cu complex decreased with time and those of the altered form of albuminCu complex increased at the expense of the former peak. Further, the latter peak eluted earlier with an increase in its intensity. When oxygen gas was removed from the reaction mixture, the change was retarded as shown in Figs. 3B and 4B. Glutathione was examined as another candidate of the hydrophilic low-molecu-
B
A :
RSentiZ
I .ospghl
tim’,” emit?
i
RSentiG
I .OS pghl
Urn’,” (mirf?
FIG. 5. Changes in Cu distribution in rat serum after an in vitro addition of cupric chloride and glutathione. Cupric chloride and the reduced form of glutathione were added in vitro to the control serum to amounts of 8 pg Cu/ml serum and 200 pg/ml serum, respectively, and then the serum solutions were incubated at 37°C for 10,30, 60, and 120 min aerobically (A) or anaerobically (B; bubbled through with 99.999% nitrogen gas and then sealed in an atmosphere of nitrogen gas). The vertical bars indicate the detector level of Cu. Peaks are assigned as indicated in the legend to Fig. 1.
576
SUZUKI, KARASAWA,
I
0 n
Retention
pg/ml
. ..l....l....l...
,....,....I...
10
5
15 time
20 (mid
10
Retentlon
15
20
time (mid
FIG. 6. Changes in sulfur distribution in rat serum after an in vitro addition of cupric chloride and glutathione. See the legend to Fig. 5. Peaks are assigned as indicated in the legend to Fig. 1.
one instead of cysteine. However, the change was much slower than that observed by the addition of cysteine, indicating that glutathione is less effective than cysteine in the conversion of the mercaptalbumin-Cu complex. As the reaction by glutathione was much slower than that by cysteine, the effect of oxygen removal on the reaction became more obvious as shown in Figs. 5B and 6B. No observable changes occurred in the distributions of Cu and sulfur and absorbances at 254 and 280 nm when the control serum was incubated with glutathione but without addition of cupric ions (data not shown). DISCUSSION
The Cu-binding site or Cu(II)-transport site has been studied for bovine and human serum albumin and its coordination structures have been proposed (10,13). Four nitrogen ligands including one imidazole, two peptides, and one amino nitrogen are involved at least to form the coordinated structure. However, sulfhydryl groups were not involved in the coordinated structure of Cu and albumin. On the other hand, some structural conversions or isomerizations of albumin such as an N-F transition or an N-A isomerization (20) and a mercapt-nonmercapt conversion (15, 21) are known to involve intra- and intermolecular conversions of sulfhydryl and sulfide groups. Especially, the mercapt-nonmercapt conversion of albumin is known to occur by the intermolecular participation of cysteine or glutathione (15,21).
AND YAMANAKA
The present observation indicated the involvement of at least three kinds of biomolecules (albumin, Cu, and cysteine) in the formation of albumin-Cu complex in viva. It is also suggested that an oxygen molecule or oxidative reaction is involved in the formation of the albumin-cu-cysteine complex. The concentration of serum glutathione is regulated by the balance of secretion from the liver and excretion by the kidneys. Glutathione excreted by the kidneys is degraded by y-glutamyltranspeptidase to yield cysteine. A small portion of cysteine liberated from glutathione in this pathway returns to the bloodstream (2224). Therefore, although cysteine is present only in a small quantity in the blood serum, it can be supplied continuously through the degradation of glutathione. This may explain the source of cysteine in the formation of the albumin-Cu-cysteine complex in viva. The overall reaction can be explained as follows: when free cupric ions are present in the blood stream, the ions bind mostly to the major form of albumin to form the mercaptalbumin-Cu complex, which sequesters cysteine to form the albumin-Cu-cysteine complex possibly in an oxidative reaction. The formation of the albumin-Cu-cysteine complex is limited by the supply of cysteine. Only Cu in the albumin-Cu-cysteine complex must be transferred to the liver with albumin remaining in the blood serum as an albumincysteine complex. The change in the distribution profile of sulfur in the albumin fraction (Fig. 1) suggests further that the albumin-cysteine complex remaining in the blood serum is then gradually reduced to form mercaptalbumin. Although an oxidative reaction between mercaptalbumin and cysteine is proposed in the formation of an albumin-cu-cysteine complex, the albumin-cysteine complex formed in the present reaction was shown to be different from nonmercaptalbumin (Fig. 1). Nonmercaptalbumin present in normal serum is an intermolecular oxidation product of albumin and cysteine or glutathione (15, 21). As albumin is known to give several structurally converted forms (26), this difference can be explained as follows. The albumin-cu-cyste-
COPPER-ALBUMIN-CYSTEINE
ine complex may be formed from a converted form of albumin which is different from the usual form of albumin, while the albumin-cysteine complex known as nonmercaptalbumin can be formed from the usual forms of albumin. Cysteine reacted more readily with the mercaptalbumin-Cu complex to form the albumin-Cu-cysteine complex than did glutathione (Figs. 3-6). This may indicate that some kind of steric hindrance causes the retardation in the formation of the albuminCu-cysteine complex with glutathione. The albumin-Cu-cysteine complex can be formed by addition of cysteine to the mercaptalbumin-Cu complex under oxidative conditions. This suggests that a sulfhydryl group participates in the formation of the albumin-Cu-cysteine complex. However, there is only one sulfhydryl group among 35 cysteinyl residues in the albumin molecule, which suggests that the intermolecular disulfide bond in the albumin-cucysteine complex may not be formed with the same sulfhydryl group as that of the usual nonmercaptalbumin. This remained unsolved in the present study. The present study revealed that Cu binds to mercaptalbumin in vivo and in vitro. Although the mercaptalbumin-Cu complex remains unchanged in vitro, it forms a complex with albumin, Cu, and cysteine in vivo and this complex can be reconstituted in vitro in the presence of cysteine or glutathione under oxidative conditions. Copper can be removed from the albumin-Cu-cysteine complex and the remaining albumin-cysteine complex can be reduced to liberate mercaptalbumin. Although the precise mechanism including its stoichiometry remains to be clarified, the in vivo reaction was reconstituted in vitro in the present study. This reaction can be detected only by a suitable separation method for albumin and also by a powerful method for simultaneous detection of Cu, sulfur, and other metals. ACKNOWLEDGMENTS The authors are grateful to Dr. E. Kobayashi for operation of the ICP spectrometer. K.T.S. and A.K. express their thanks to Dr. M. Murakami for his en-
COMPLEX
IN RAT SERUM
577
couragement. This work is partly supported by the Human Science Foundation (Code No. 3-3-l-A). REFERENCES 1. LAW, S.-J., AND SARKAR, B. (1984) Canad J. Biothem. Cell. Biol62,449-455. 2. LAUSSAC, J.-P., AND SARKAR, B. (1984) Biochemistry 23,2832-2838. 3. MARTINS, E. O., AND DRAKENBERG, T. (1982) Inorg. Chim Acta 67,71-74. 4. MASON, K. E. (1979) J. Nutr. 109,19’79-2066. 5. DAWSON, J. B., BAHREYNI-TOOSI, M. H., ELLIS, D. J., AND HODGKINSON, A. (1981) Analyst 106, 153-159. 6. WEISS, K. C., AND LINDER, M. (1985) Amer. J. Physiol. 249, E77-E88. 7. OWEN, C. A., JR. (1965) Amer. J. Physiol209, SOO964. 8. RYDEN, L., AND BJORK, I. (1976) Biochemistry 15, 3411-3417. 9. PETERS, T., JR., AND HAWN, C. (1967) J. Biol. Chem 242,1566-1573. 10. PETERS, T., JR., AND BLUMENSTOCK, F. A. (1967) J. Biol Chem. 242,1574-1578. 11. BRADSHAW, R. A., AND PETERS, T., JR. (1969) J. Biol. Chem. 244,5582-5589. 12. BRADSHAW, R. A., SHEARER, W. T., AND GURD, F. R. N. (1968) J. Biol. Chem. 243,3817-3825. 13. LAUSSAC, J.-P., AND SARKAR, B. (1980) J. Biol. Chem. 255,7563-7568. 14. SOGAMI, M., YANG, J. T., AND AOKI, K., (Eds.) (1980) Selected Papers on Plasma Albumin: Some Aspects of Its Structure and Conformation. pp. l-409. 15. SOGAMI, M., ERA, S., NAGAOKA, S., KUWATA, K., KIDA, K., AND SHIGEMI, J. (1985) J. Chromatogr. 332,19-27. 16. SUZUKI, K. T., SUNAGA, H., KOBAYASHI, E., AND SHIMOJO, N. (1986) Toxicol. Appl. Pharmacol 86,466-473. 17. SUNAGA, H., KOBAYASHI, E., SHIMOJO, N., AND SUZUKI, K. T. (1987) Anal Biochem 160,160168. 18. SUZUKI, K. T., SUNAGA, H., KOBAYASHI, E., AND SUGIHIRA, N. (1987) J. Chromdogr. 400,233-240. 19. SUZUKI, K. T., KARASAWA, A., SUNAGA, H., KoDAMA, H., AND YAMANAKA, K. (1989) Camp. Biochem PhysioL, in press. 20. FOSTER, J. F. (1977) in The Albumin Structure, Function and Uses (Rosenoer, V. M., Oratz, M., and Rothshild, M. A., Eds.), pp. 53-84, Pergamon Press, Oxford. 21. SOGAMI, M., NAGAOKA, S., ERA, S., HONDA, M., AND NOGUCHI, N. (1984) Int. J. Peptide Protein Res. 24,96-103. 22. CURTHOYS, N. P. (1988) Miner. Electrolyte Me&b. 9,236-245. 23. HILL, K. E., VON HOFF, D. D., AND BURK, R. F. (1985) Invest. New Drugs 3,31-34. 24. LASH, L. H., AND JONES, D. P. (1985) Arch Bb them. Biophys. 240,583-592.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 2’73, No. 2, September, pp. 572-5’77,1989
Binding of Copper to Albumin and Participation of Cysteine in Vivo and in Vitro KAZUO T. SUZUKI,**1 AKIRA
KARASAWA,**t
AND
KEI YAMANAKAT
*National Institute for Environmental Studies, 0nogawa, Tsukuba, I&-ski 305, Japan, and ~Institute of Environmental Sciences, University of Tsukuba, Amakubo, Tsukuba 305, Japan Received April 7,1989, and in revised form May 22,1989
Albumin, the major copper-binding protein in blood serum, was shown to form different albumin-copper complexes in in vivo and in vitro. Cupric ions added in vitro to control rat serum bound preferentially to mercaptalbumin and the mercaptalbumin-copper complex remained unchanged with time. Cupric ions injected intravenously into the rat first formed the mercaptalbumin-copper complex; this binary complex changed gradually with time to form an albumin-copper-cysteine complex. The participation of cysteine in the formation of this complex was demonstrated in vitro and further suggested that its conversion was an oxidative reaction. Glutathione also participated in forming the complex, but it was not as effective as cysteine. Albumin-copper complexes were separated on a gel filtration column and detected simultaneously by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry. 0 1989 Academic Press. Inc.
Albumin is a major binding protein for metal ions in blood serum and several binding sites have already been characterized for some metals (l-3). Copper is one of the metals that bind to albumin in the blood. Over 90% of the Cu in human plasma is firmly bound to an az-globulin, ceruloplasmin, and the remainder is less firmly bound in large part to albumin (4, 5). However, this does not mean that ceruloplasmin is the major carrier protein for the transport of Cu absorbed in the blood plasma (6). Copper in ceruloplasmin is incorporated during its synthesis in the liver (7) and Cu bound to ceruloplasmin does not exchange or equilibrate with ionic Cu in vitro (8). Therefore, although Cu bound to albumin is less than 10% of plasma Cu, albumin is probably the largest labile pool for plasma Cu and may be the most important Cu carrier component in blood serum (4). The Cu(II)-transport site of serum albumin has been extensively studied and has
1 To whom correspondence
should be addressed.
0003-9861/89 $3.00 Copyright All rights
0 1989 hy Academic Press, Inc. of reproduction in any form resewed.
been suggested to involve the a-amino nitrogen, two intervening peptide nitrogens, and the imidazole nitrogen of the histidine residue in position 3 (9-13). Extensive investigations in the Foster’s laboratory contributed to clarify the structure and conformation of plasma albumin as compiled in “Selected Papers on Plasma Albumin” by his students (14). Plasma albumin is a mixture of mercaptalbumin and nonmercaptalbumin, the two forms of human albumin have been separated on a high-performance liquid chromatograph using an Asahipak GS-520 column (15). Mercaptalbumin and nonmercaptalbumin in rat plasma have been separated on the same column under different conditions and mercaptalbumin was identified as the serum carrier for Cd (16). Metals bound firmly to specific binding sites of metalloproteins can be separated without dissociation from the proteins during separation procedures. However, metals bound less firmly to proteins may interact with ligands of column resins, resulting in redistribution or dissociation of 572
COPPER-ALBUMIN-CYSTEINE
COMPLEX IN RAT SERUM
573
metal ions. Silanol groups in silica gels when 12 weeks old (body weight, approximately 290 g). Cupric chloride in physiological saline was inmay work as efficient anionic counterparts of metallic cations and gel filtration col- jected into the femoral vein (three to five rats per umns without silica gels are preferable for group) as a singledoseof 0.8 mgCu/kg body wt under anesthesia. The rats were killed 5, 10, the separation of metal-binding proteins. pentobarbital 60, and 180 min later by exsanguination. The blood An Asahipak GS-520 column used for sep- was centrifuged at 23OOgfor 10 min and the serum aration of mercaptalbumin and nonmer- was analyzed immediately by the HPLC-ICP method captalbumin is made of resins without sil- (16-18). Control serum was prepared from rats withica gels and can be effectively used to sepa- out any treatment. rate metal-binding proteins (16). In vitro treatments. A series of experiments were Biological samples contain a variety of carried out using fresh control serum each time. Cupric chloride and/or cysteine were added to the serum elements and these elements interact with to concentrations of 8 pg Cu/ml serum and/or 80 pg each other in the body. Hence, the simultacysteine/ml serum. The serum was incubated at 37°C neous detection of multi-elements is someaerobically without sealing the test tube or anaerobitimes essential to understand biological cally with sealing the test tube in an atmosphere of reactions in which metal ions are involved. nitrogen gas. We have developed an efficient analytical Analysis bg the HPLC-ICP method (16-18). A &l-ml method for the simultaneous determinaportion of serum was applied to an Asahipak GS-520 tion of elements in biological samples; an column (7.6 X 500 mm, Asahi Chemical Co. Ltd., Toinductively coupled argon plasma-atomic kyo) and the column was eluted with 10 mM Tris/HCl emission spectrometer has been used buffer solution (pH 7.4, containing 0.9% NaCl and as a multi-element-specific detector with 0.05% NaNa, the dissolved gases were removed before use with a degasser) at a flow rate of 1.0 ml/min on HPLC (16-18). During analysis of Cu in blood serum, we an HPLC (Model 340, Beckman). Concentrations of multi-elements in the eluate were determined simulobserved that Cu-albumin complexes taneously using an ICP spectrometer (JY48 PVH, formed in viva and in vitro show different Seiko Instruments & Electronics, Ltd., Tokyo) as a decharacteristics on a gel filtration column tector of HPLC. Absorbances at 254 and 280 nm were by the above HPLC-ICP’ method. Cupric monitored with a dual-wavelength uv detector (Model ions added in vitro to rat serum were bound 152, Altex, Berkeley). The distribution profiles were preferentially to mercaptalbumin and the drawn using a personal computer (PC-9801, NEC Cu-mercaptalbumin complex remained Corp., Tokyo) and an XY-plotter (FP5301R, Graphtec unchanged with time. On the other hand, Corp., Tokyo). the Cu-albumin complex formed in vivo changed with time (19). This difference beRESULTS tween in viva and in vitro reactions was obUnder the present column conditions, served only when mercaptalbumin and nonmercaptalbumin were separated, and serum proteins were largely separated into when Cu and albumin forms were sepa- globulins (eluted before 15.0 min) and alrated as metal-binding complexes. The bumins (eluted after 15.6 min). Sulfur was metal and proteins have to be detected si- detected with an ultravacuum ICP specmultaneously for this purpose. The present trometer as shown in Fig. 1B. The distribustudy was undertaken to clarify the con- tion of sulfur can be used as an efficient proteins tributing factors in the formation of tool to detect sulfur-containing different Cu-albumin complexes between and low-molecular-weight compounds; the in viva and in vitro systems utilizing the highest sulfur peak at 15.2 min in the control profile in Fig. 1 was assigned to merHPLC-ICP method. captalbumin, while a broad shoulder peak MATERIALS AND METHODS at around 19.0 min was assigned to nonmercaptalbumin (16). The shoulder peak at Animals Female rats of the Wiatar strain were 17.0 min and a peak at 18.1 min in the conpurchased from Clea Japan Co., Tokyo, and were used trol sulfur profile were due to sulfate ions and glutathione, respectively (checked by using appropriate standards). The sulfur x Abbreviation used: ICP, inductiveiy coupled argon distribution can be used as a comparable plasma-atomic emission spectrometry,
574
SUZUKI,
KARASAWA,
B 5 pg/ml
A n I
I....I....I....I
10
..l....l....‘....
15
Retention
20 time
25 (mid
10 Retention
15
20 time
(mid
FIG. 1. Changes in Cu and sulfur distributions in rat serum after a single intravenous injection of cupric chloride. A and B show the distribution profiles of Cu and sulfur, respectively, in rat serum. The animals were injected intravenously with CuCla at a dose of 0.8 mg Cu/kg and were killed 5 and 10 min and 1 and 3 h later. The vertical bars indicate the detection level of Cu or sulfur by the HPLC-ICP method. Peaks are assigned as follows: ceruloplasmin (12.7 min in A), mercaptalbumin (15.2 min in A and B), sulfate ion (17.0 min in B), glutathione (18.1 min in B), nonmercaptalbumin (19.0 min in A and B).
or superior tool to the absorbances at 254 and 280 nm (Fig. 2B). Copper in rat serum was bound mostly to ceruloplasmin (retention time 12.7 min) and only in a small amount to albumin (15.3 min) as shown in Fig. 1. However, cupric ions injected intravenously into the rat did not distribute to ceruloplasmin and was bound mostly to albumin. Copper distributed to albumin was eluted separately into two peaks; one was eluted at 15.2 min (at the position of mercaptalbumin) and the other at 17.0 min (at the position between mercaptalbumin and nonmercaptalbumin) (Fig. lA, 5 min). The relative intensities of these two Cu peaks changed with time, while the intensity of ceruloplasmin remained constant. In accordance with the change in Cu distribution in the albumin fraction., sulfur distribution in the albumin fraction also changed dramatically. However, the sulfur distribution in the globulin fraction was not altered (Fig. 1B). When the sulfur distribution in the albumin fraction was examined precisely, the change in sulfur distribution can be summarized as follows: mercaptalbumin was converted rapidly with time to a different form of al-
AND
YAMANAKA
bumin in the presence of Cu and the altered form of albumin started to return to the original mercaptalbumin when Cu disappeared from the blood serum. Although the distributions of Cu and sulfur and absorbances at 254 and 280 nm (data not shown) in the albumin fraction changed with time after injection of cupric ion into the rat, these changes were not observed when cupric ions were added in witro into the control serum as shown in Fig. 2. Copper was bound to mercaptalbumin and the albumin peaks as monitored by the distribution profiles of sulfur and absorbances at 254 and 280 nm remained unchanged. The mercaptalbumin-Cu complex first formed in viva was assumed to be converted into an altered form of albumin-Cu complex. The altered form of albuminCu complex was eluted slower than the mercaptalbumin-Cu complex and faster than the nonmercaptalbumin-Cu complex, which suggested the participation of a hydrophilic low-molecular-weight compound in the rat serum in this conversion. Cysteine was examined as a candidate comnound in the blood serum, as shown in Fig. 3.
g-fJe 382 1 Omin
30min
FIG. 2. Changes in Cu and sulfur distributions in rat serum after an in vitro addition of cupric chloride. Cupric chloride was added to the control serum to an amount of 8 Irg Cu/ml serum and then incubated at 37°C for 10 or 30 min. A shows the distribution profiles of Cu, while the upper two profiles and lower three profiles in B show the distribution profiles of absorbances at 254 and 280 nm, and sulfur for the control, respectively. The vertical bars indicate the detection level of Cu or sulfur. Peaks are assigned as indicated in the legend to Fig. 1.
COPPER-ALBUMIN-CYSTEINE
IN RAT
lJ$&
AL
Omin
min
Omin
1Omin
Lmin
lJdL
1 Omin
Retention
25
time (mid
RZentiZ-4
tin?: (mi3
FIG. 3. Changes in Cu distribution in rat serum after an in vitro addition of cupric chloride and cysteine. Cupric chloride and cysteine were added in vitro to the control serum to amounts of 8 pg Cu/ml serum and 80 pg/ml serum, respectively, and then the serum solutions were incubated at 37°C for 5,10,20, and 30 min aerobically (A) or anaerobically (B; bubbled through with 99.999% nitrogen gas and then sealed in an atmosphere of nitrogen gas). Peaks are assigned as indicated in the legend to Fig. 1.
I....,,..,,,,
15
Retention
II
20
Smin
1OWl
i/-L
I....,....,..,
30min 15
skb& Omin
30min
10 20min
10
I 5 pa/ml
B
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A
575
SERUM
5 fig/ml
I. 05 ye/ml
CU
COMPLEX
20
time
(mid
10
15
Retention
20
time
(min)
FIG. 4. Changes in sulfur distribution in rat serum after an in vitro addition of cupric chloride and cysteine. See the legend to Fig. 3. The vertical bars indicate the detector level of sulfur. Peaks are assigned as indicated in the legend to Fig. 1.
lar-weight compound in the serum, as shown in Figs. 5 and 6. Similar changes in the distributions of Cu and sulfur were also observed by the addition of glutathi-
Cupric ions and cysteine were added in vitro into the control serum and distribu-
tions of Cu and sulfur were determined by the HPLC-ICP method after incubation for 5, 10, 20, and 30 min. Although dissolved oxygen gas might not be removed completely, the incubation was also carried out in an atmosphere of nitrogen gas after bubbling through with nitrogen gas. Copper added in vitro without cysteine remained unchanged as mercaptalbumin-Cu complex even after 30 min (Fig. 2). However, an addition of cysteine into the control serum along with cupric ions induced changes in the distributions of both Cu and sulfur as shown in Figs. 3 and 4. The intensities of Cu and sulfur peaks of the mercaptalbumin-Cu complex decreased with time and those of the altered form of albuminCu complex increased at the expense of the former peak. Further, the latter peak eluted earlier with an increase in its intensity. When oxygen gas was removed from the reaction mixture, the change was retarded as shown in Figs. 3B and 4B. Glutathione was examined as another candidate of the hydrophilic low-molecu-
B
A :
RSentiZ
I .ospghl
tim’,” emit?
i
RSentiG
I .OS pghl
Urn’,” (mirf?
FIG. 5. Changes in Cu distribution in rat serum after an in vitro addition of cupric chloride and glutathione. Cupric chloride and the reduced form of glutathione were added in vitro to the control serum to amounts of 8 pg Cu/ml serum and 200 pg/ml serum, respectively, and then the serum solutions were incubated at 37°C for 10,30, 60, and 120 min aerobically (A) or anaerobically (B; bubbled through with 99.999% nitrogen gas and then sealed in an atmosphere of nitrogen gas). The vertical bars indicate the detector level of Cu. Peaks are assigned as indicated in the legend to Fig. 1.
576
SUZUKI, KARASAWA,
I
0 n
Retention
pg/ml
. ..l....l....l...
,....,....I...
10
5
15 time
20 (mid
10
Retentlon
15
20
time (mid
FIG. 6. Changes in sulfur distribution in rat serum after an in vitro addition of cupric chloride and glutathione. See the legend to Fig. 5. Peaks are assigned as indicated in the legend to Fig. 1.
one instead of cysteine. However, the change was much slower than that observed by the addition of cysteine, indicating that glutathione is less effective than cysteine in the conversion of the mercaptalbumin-Cu complex. As the reaction by glutathione was much slower than that by cysteine, the effect of oxygen removal on the reaction became more obvious as shown in Figs. 5B and 6B. No observable changes occurred in the distributions of Cu and sulfur and absorbances at 254 and 280 nm when the control serum was incubated with glutathione but without addition of cupric ions (data not shown). DISCUSSION
The Cu-binding site or Cu(II)-transport site has been studied for bovine and human serum albumin and its coordination structures have been proposed (10,13). Four nitrogen ligands including one imidazole, two peptides, and one amino nitrogen are involved at least to form the coordinated structure. However, sulfhydryl groups were not involved in the coordinated structure of Cu and albumin. On the other hand, some structural conversions or isomerizations of albumin such as an N-F transition or an N-A isomerization (20) and a mercapt-nonmercapt conversion (15, 21) are known to involve intra- and intermolecular conversions of sulfhydryl and sulfide groups. Especially, the mercapt-nonmercapt conversion of albumin is known to occur by the intermolecular participation of cysteine or glutathione (15,21).
AND YAMANAKA
The present observation indicated the involvement of at least three kinds of biomolecules (albumin, Cu, and cysteine) in the formation of albumin-Cu complex in viva. It is also suggested that an oxygen molecule or oxidative reaction is involved in the formation of the albumin-cu-cysteine complex. The concentration of serum glutathione is regulated by the balance of secretion from the liver and excretion by the kidneys. Glutathione excreted by the kidneys is degraded by y-glutamyltranspeptidase to yield cysteine. A small portion of cysteine liberated from glutathione in this pathway returns to the bloodstream (2224). Therefore, although cysteine is present only in a small quantity in the blood serum, it can be supplied continuously through the degradation of glutathione. This may explain the source of cysteine in the formation of the albumin-Cu-cysteine complex in viva. The overall reaction can be explained as follows: when free cupric ions are present in the blood stream, the ions bind mostly to the major form of albumin to form the mercaptalbumin-Cu complex, which sequesters cysteine to form the albumin-Cu-cysteine complex possibly in an oxidative reaction. The formation of the albumin-Cu-cysteine complex is limited by the supply of cysteine. Only Cu in the albumin-Cu-cysteine complex must be transferred to the liver with albumin remaining in the blood serum as an albumincysteine complex. The change in the distribution profile of sulfur in the albumin fraction (Fig. 1) suggests further that the albumin-cysteine complex remaining in the blood serum is then gradually reduced to form mercaptalbumin. Although an oxidative reaction between mercaptalbumin and cysteine is proposed in the formation of an albumin-cu-cysteine complex, the albumin-cysteine complex formed in the present reaction was shown to be different from nonmercaptalbumin (Fig. 1). Nonmercaptalbumin present in normal serum is an intermolecular oxidation product of albumin and cysteine or glutathione (15, 21). As albumin is known to give several structurally converted forms (26), this difference can be explained as follows. The albumin-cu-cyste-
COPPER-ALBUMIN-CYSTEINE
ine complex may be formed from a converted form of albumin which is different from the usual form of albumin, while the albumin-cysteine complex known as nonmercaptalbumin can be formed from the usual forms of albumin. Cysteine reacted more readily with the mercaptalbumin-Cu complex to form the albumin-Cu-cysteine complex than did glutathione (Figs. 3-6). This may indicate that some kind of steric hindrance causes the retardation in the formation of the albuminCu-cysteine complex with glutathione. The albumin-Cu-cysteine complex can be formed by addition of cysteine to the mercaptalbumin-Cu complex under oxidative conditions. This suggests that a sulfhydryl group participates in the formation of the albumin-Cu-cysteine complex. However, there is only one sulfhydryl group among 35 cysteinyl residues in the albumin molecule, which suggests that the intermolecular disulfide bond in the albumin-cucysteine complex may not be formed with the same sulfhydryl group as that of the usual nonmercaptalbumin. This remained unsolved in the present study. The present study revealed that Cu binds to mercaptalbumin in vivo and in vitro. Although the mercaptalbumin-Cu complex remains unchanged in vitro, it forms a complex with albumin, Cu, and cysteine in vivo and this complex can be reconstituted in vitro in the presence of cysteine or glutathione under oxidative conditions. Copper can be removed from the albumin-Cu-cysteine complex and the remaining albumin-cysteine complex can be reduced to liberate mercaptalbumin. Although the precise mechanism including its stoichiometry remains to be clarified, the in vivo reaction was reconstituted in vitro in the present study. This reaction can be detected only by a suitable separation method for albumin and also by a powerful method for simultaneous detection of Cu, sulfur, and other metals. ACKNOWLEDGMENTS The authors are grateful to Dr. E. Kobayashi for operation of the ICP spectrometer. K.T.S. and A.K. express their thanks to Dr. M. Murakami for his en-
COMPLEX
IN RAT SERUM
577
couragement. This work is partly supported by the Human Science Foundation (Code No. 3-3-l-A). REFERENCES 1. LAW, S.-J., AND SARKAR, B. (1984) Canad J. Biothem. Cell. Biol62,449-455. 2. LAUSSAC, J.-P., AND SARKAR, B. (1984) Biochemistry 23,2832-2838. 3. MARTINS, E. O., AND DRAKENBERG, T. (1982) Inorg. Chim Acta 67,71-74. 4. MASON, K. E. (1979) J. Nutr. 109,19’79-2066. 5. DAWSON, J. B., BAHREYNI-TOOSI, M. H., ELLIS, D. J., AND HODGKINSON, A. (1981) Analyst 106, 153-159. 6. WEISS, K. C., AND LINDER, M. (1985) Amer. J. Physiol. 249, E77-E88. 7. OWEN, C. A., JR. (1965) Amer. J. Physiol209, SOO964. 8. RYDEN, L., AND BJORK, I. (1976) Biochemistry 15, 3411-3417. 9. PETERS, T., JR., AND HAWN, C. (1967) J. Biol. Chem 242,1566-1573. 10. PETERS, T., JR., AND BLUMENSTOCK, F. A. (1967) J. Biol Chem. 242,1574-1578. 11. BRADSHAW, R. A., AND PETERS, T., JR. (1969) J. Biol. Chem. 244,5582-5589. 12. BRADSHAW, R. A., SHEARER, W. T., AND GURD, F. R. N. (1968) J. Biol. Chem. 243,3817-3825. 13. LAUSSAC, J.-P., AND SARKAR, B. (1980) J. Biol. Chem. 255,7563-7568. 14. SOGAMI, M., YANG, J. T., AND AOKI, K., (Eds.) (1980) Selected Papers on Plasma Albumin: Some Aspects of Its Structure and Conformation. pp. l-409. 15. SOGAMI, M., ERA, S., NAGAOKA, S., KUWATA, K., KIDA, K., AND SHIGEMI, J. (1985) J. Chromatogr. 332,19-27. 16. SUZUKI, K. T., SUNAGA, H., KOBAYASHI, E., AND SHIMOJO, N. (1986) Toxicol. Appl. Pharmacol 86,466-473. 17. SUNAGA, H., KOBAYASHI, E., SHIMOJO, N., AND SUZUKI, K. T. (1987) Anal Biochem 160,160168. 18. SUZUKI, K. T., SUNAGA, H., KOBAYASHI, E., AND SUGIHIRA, N. (1987) J. Chromdogr. 400,233-240. 19. SUZUKI, K. T., KARASAWA, A., SUNAGA, H., KoDAMA, H., AND YAMANAKA, K. (1989) Camp. Biochem PhysioL, in press. 20. FOSTER, J. F. (1977) in The Albumin Structure, Function and Uses (Rosenoer, V. M., Oratz, M., and Rothshild, M. A., Eds.), pp. 53-84, Pergamon Press, Oxford. 21. SOGAMI, M., NAGAOKA, S., ERA, S., HONDA, M., AND NOGUCHI, N. (1984) Int. J. Peptide Protein Res. 24,96-103. 22. CURTHOYS, N. P. (1988) Miner. Electrolyte Me&b. 9,236-245. 23. HILL, K. E., VON HOFF, D. D., AND BURK, R. F. (1985) Invest. New Drugs 3,31-34. 24. LASH, L. H., AND JONES, D. P. (1985) Arch Bb them. Biophys. 240,583-592.