Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 Nov;7(11):2398–2404. doi: 10.1002/pro.5560071117

Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometry.

J Zaia 1, D Fabris 1, D Wei 1, R L Karpel 1, C Fenselau 1
PMCID: PMC2143854  PMID: 9828006

Abstract

The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.

Full Text

The Full Text of this article is available as a PDF (733.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bittner M., Burke R. L., Alberts B. M. Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities. J Biol Chem. 1979 Oct 10;254(19):9565–9572. [PubMed] [Google Scholar]
  2. Brader M. L., Dunn M. F. Insulin hexamers: new conformations and applications. Trends Biochem Sci. 1991 Sep;16(9):341–345. doi: 10.1016/0968-0004(91)90140-q. [DOI] [PubMed] [Google Scholar]
  3. Czupryn M., Brown W. E., Vallee B. L. Zinc rapidly induces a metal response element-binding factor. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10395–10399. doi: 10.1073/pnas.89.21.10395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gan T., Munoz A., Shaw C. F., 3rd, Petering D. H. Reaction of 111Cd7-metallothionein with EDTA. A reappraisal. J Biol Chem. 1995 Mar 10;270(10):5339–5345. doi: 10.1074/jbc.270.10.5339. [DOI] [PubMed] [Google Scholar]
  5. Giedroc D. P., Keating K. M., Williams K. R., Konigsberg W. H., Coleman J. E. Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8452–8456. doi: 10.1073/pnas.83.22.8452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hao R., Pfeiffer R. F., Ebadi M. Purification and characterization of metallothionein and its activation of pyridoxal phosphokinase in trout (Salmo gairdneri) brain. Comp Biochem Physiol B. 1993 Feb;104(2):293–298. doi: 10.1016/0305-0491(93)90371-b. [DOI] [PubMed] [Google Scholar]
  7. Jensen D. E., Kelly R. C., von Hippel P. H. DNA "melting" proteins. II. Effects of bacteriophage T4 gene 32-protein binding on the conformation and stability of nucleic acid structures. J Biol Chem. 1976 Nov 25;251(22):7215–7228. [PubMed] [Google Scholar]
  8. Kelley S. L., Basu A., Teicher B. A., Hacker M. P., Hamer D. H., Lazo J. S. Overexpression of metallothionein confers resistance to anticancer drugs. Science. 1988 Sep 30;241(4874):1813–1815. doi: 10.1126/science.3175622. [DOI] [PubMed] [Google Scholar]
  9. Lazo J. S., Kondo Y., Dellapiazza D., Michalska A. E., Choo K. H., Pitt B. R. Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes. J Biol Chem. 1995 Mar 10;270(10):5506–5510. doi: 10.1074/jbc.270.10.5506. [DOI] [PubMed] [Google Scholar]
  10. Loo J. A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev. 1997 Jan-Feb;16(1):1–23. doi: 10.1002/(SICI)1098-2787(1997)16:1<1::AID-MAS1>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  11. Maret W., Larsen K. S., Vallee B. L. Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2233–2237. doi: 10.1073/pnas.94.6.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Maret W. Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem Int. 1995 Jul;27(1):111–117. doi: 10.1016/0197-0186(94)00173-r. [DOI] [PubMed] [Google Scholar]
  13. Maret W. Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):237–241. doi: 10.1073/pnas.91.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Masters B. A., Kelly E. J., Quaife C. J., Brinster R. L., Palmiter R. D. Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):584–588. doi: 10.1073/pnas.91.2.584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Michalska A. E., Choo K. H. Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8088–8092. doi: 10.1073/pnas.90.17.8088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nicholson J. K., Sadler P. J., Vasák M. Probing the reactivity of the zinc and cadmium ions bound to rabbit liver metallothioneins with EDTA. Experientia Suppl. 1987;52:191–201. doi: 10.1007/978-3-0348-6784-9_12. [DOI] [PubMed] [Google Scholar]
  17. Nielson K. B., Winge D. R. Order of metal binding in metallothionein. J Biol Chem. 1983 Nov 10;258(21):13063–13069. [PubMed] [Google Scholar]
  18. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411–2423. doi: 10.1002/pro.5560041120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Philcox J. C., Coyle P., Michalska A., Choo K. H., Rofe A. M. Endotoxin-induced inflammation does not cause hepatic zinc accumulation in mice lacking metallothionein gene expression. Biochem J. 1995 Jun 1;308(Pt 2):543–546. doi: 10.1042/bj3080543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Robbins A. H., McRee D. E., Williamson M., Collett S. A., Xuong N. H., Furey W. F., Wang B. C., Stout C. D. Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution. J Mol Biol. 1991 Oct 20;221(4):1269–1293. [PubMed] [Google Scholar]
  21. Rofe A. M., Philcox J. C., Coyle P. Trace metal, acute phase and metabolic response to endotoxin in metallothionein-null mice. Biochem J. 1996 Mar 15;314(Pt 3):793–797. doi: 10.1042/bj3140793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Savas M. M., Shaw C. F., 3rd, Petering D. H. The oxidation of rabbit liver metallothionein-II by 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfide. J Inorg Biochem. 1993 Dec;52(4):235–249. doi: 10.1016/0162-0134(93)80028-8. [DOI] [PubMed] [Google Scholar]
  23. Schultze P., Wörgötter E., Braun W., Wagner G., Vasák M., Kägi J. H., Wüthrich K. Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy. J Mol Biol. 1988 Sep 5;203(1):251–268. doi: 10.1016/0022-2836(88)90106-4. [DOI] [PubMed] [Google Scholar]
  24. Shamoo Y., Adari H., Konigsberg W. H., Williams K. R., Chase J. W. Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8844–8848. doi: 10.1073/pnas.83.23.8844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shamoo Y., Webster K. R., Williams K. R., Konigsberg W. H. A retrovirus-like zinc domain is essential for translational repression of bacteriophage T4 gene 32. J Biol Chem. 1991 May 5;266(13):7967–7970. [PubMed] [Google Scholar]
  26. Thumann J., Grill E., Winnacker E. L., Zenk M. H. Reactivation of metal-requiring apoenzymes by phytochelatin-metal complexes. FEBS Lett. 1991 Jun 17;284(1):66–69. doi: 10.1016/0014-5793(91)80763-s. [DOI] [PubMed] [Google Scholar]
  27. Udom A. O., Brady F. O. Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thionein. Biochem J. 1980 May 1;187(2):329–335. doi: 10.1042/bj1870329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vallee B. L. The function of metallothionein. Neurochem Int. 1995 Jul;27(1):23–33. doi: 10.1016/0197-0186(94)00165-q. [DOI] [PubMed] [Google Scholar]
  29. Vasák M., Wörgötter E., Wagner G., Kägi J. H., Wüthrich K. Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2. J Mol Biol. 1987 Aug 5;196(3):711–719. doi: 10.1016/0022-2836(87)90042-8. [DOI] [PubMed] [Google Scholar]
  30. Vazquez F., Vasák M. Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a. Biochem J. 1988 Jul 15;253(2):611–614. doi: 10.1042/bj2530611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Williams K. R., LoPresti M. B., Setoguchi M. Primary structure of the bacteriophage T4 DNA helix-destabilizing protein. J Biol Chem. 1981 Feb 25;256(4):1754–1762. [PubMed] [Google Scholar]
  32. Yu X., Wojciechowski M., Fenselau C. Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometry. Anal Chem. 1993 May 15;65(10):1355–1359. doi: 10.1021/ac00058a010. [DOI] [PubMed] [Google Scholar]
  33. Yu X., Wu Z., Fenselau C. Covalent sequestration of melphalan by metallothionein and selective alkylation of cysteines. Biochemistry. 1995 Mar 14;34(10):3377–3385. doi: 10.1021/bi00010a029. [DOI] [PubMed] [Google Scholar]
  34. Zaia J., Jiang L., Han M. S., Tabb J. R., Wu Z., Fabris D., Fenselau C. A binding site for chlorambucil on metallothionein. Biochemistry. 1996 Mar 5;35(9):2830–2835. doi: 10.1021/bi952243n. [DOI] [PubMed] [Google Scholar]
  35. Zeng J., Vallee B. L., Kägi J. H. Zinc transfer from transcription factor IIIA fingers to thionein clusters. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9984–9988. doi: 10.1073/pnas.88.22.9984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES