[49]
AMINO ACID SEQUENCE DETERMINATION
421
with 50/tl of 30% H202 (Perhydrol, Fluka, Switzerland) and incubated at room temperature for 2 hr. Only freshly prepared reagent should be used. Oxidation of Salt-Free Samples. Depending on the analysis system employed, appropriate amounts of MT are dried in a hydrolysis tube, dissolved in 25- 50/d distilled formic acid, chilled, and mixed with 50100/d of cold performic acid (the final ratio of formic acid to performic acid is 1 : 2). After incubation for 3 hr on ice 25- 50 pl of water is added and the sample is dried in a Speed Vac concentrator (Savant, Hicksville, NY) or freeze-dried and hydrolyzed.
Acid Hydrolysis Oxidized or alkylated salt-free MT is hydrolyzed for 22 hr at 110 ° in vacuo or under argon in 6 M HC1. Recoveries of threonine and serine may be calculated by extrapolation to zero time after hydrolysis for 16, 40, and 72 hr.
Amino Acid Analysis of Acid Hydrolysate Normally, acid hydrolysates of MT are applied to a commercially available amino acid analyzer. With a Durrum D-500 amino acid analyzer the optimal amount of hydrolyzed protein for analysis is approximately 10 or 0.5 /tg using detection with ninhydrin or o-phthalaldehyde reagent, respectively. With analyzers from other manufacturers (model 420A, Applied Biosystems, Inc., Foster City, CA; or Amino Quant, Hewlett-Packard) accurate results are obtained using approximately 50 ng of sample.
[49] Amino Acid Sequence
Determination
By PETER E. HUNZIKER Introduction Free cysteine residues are usually not detectable as phenylthiohydantoin (PTH) derivatives obtained by Edman degradation. Because this amino acid represents up to 33% of all amino acid residues in metallothioneins (MTs), it must be chemically modified for the unambiguous determination of the primary structure of this protein. In addition to the variety of cysteine derivatives employed in peptide mappingm and sequence W. R. Berhnard, M. Va~k, and J. H. R. KAgi, Biochemistry 25, 1975 (1986).
METHODS IN ENZYMOLOGY, VOL. 205
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
422
CHEMICAL CHARACTERIZATION OF METALLOTHIONEINS
[49]
analysis, 3-7 good results were obtained in our laboratory using the S-methylated derivative of MT. 8 Complete primary structure of MT is achieved by N-terminal sequence analysis of the modified protein and by sequence analysis of peptides derived from a number of proteolytic digests. In this chapter the chemical and enzymatic fragmentations usually necessary for complete determination of the amino acid sequence of this protein are described. Material and Methods
Sequencing Equipment Gas-phase sequencer, model 470A/900A, equipped with an online PTH analyzer, model 120A (Applied Biosystems, Inc., Foster City, CA) Pulsed-liquid phase sequencer, model 477A, equipped with an online PTH analyzer, model 120A (Applied Biosystems, Inc.)
Chemicals Chemicals for sequence analysis: All are purchased from Applied Biosystems, Inc. Proteolytic enzymes ("sequencing grade"): All are supplied by Boehringer (Mannheim, Germany) and reconstituted according to instructions Trifluoroacetic acid (TFA) for high-performance liquid chromatography (HPLC) is obtained from Pierce (Rockford, IL) All other chemicals: All are of analytical or HPLC grade and are supplied by Fluka (Bucks, Switzerland) or by Merck Cysteine Modification. Purified apoMTs are modified by S-methylation prior to peptide mapping and sequence analysis, s,9 Acid Cleavage. This procedure is necessary only for vertebrate MTs containing an acetylated N terminus. Up to 1 nmol of S-methylated MT is 2 K. Mfinger and K. Lerch, Inorg. Chim. Acta 151, 11 (1988). 3 T. Kojima, C. Berger, and J. H. R. K~gi, in "Metallothionein" (J. H. R. K~gi and M. Nordberg, eds.), p. 153. Birlda~user, Basel, 1979. 4 K. M~nger, U. A. Germann, M. Beltramini, D. Niedermann, G. Baitella, J. H. R. K~gi, and K. Lerch, J. Biol. Chem. 260, 10032 (1985). 5 I.-Y. Huang, A. Yoshida, H. Tsunoo, and H. Nakajima, J. Biol. Chem. 252, 8217 (1977). 6 I.-Y. Huang, M. Kimura, A. Hata, H. Tsunoo, and A. Yoshida, J. Biochem. 89, 1839 (1981). 7D. R. Winge, K. B. Nielson, R. D. Zeikus, and W. R. Gray, J. Biol. Chem. 259, 11419 (1984). 8 p. E. Hunziker, this volume [45]. 9 p. E. Hunziker, this volume [53].
[49]
AMINO ACID SEQUENCE DETERMINATION
MT-2B
423
MT-2A
o~
6
AMINO ACID SEQUENCE DETERMINATION
421
with 50/tl of 30% H202 (Perhydrol, Fluka, Switzerland) and incubated at room temperature for 2 hr. Only freshly prepared reagent should be used. Oxidation of Salt-Free Samples. Depending on the analysis system employed, appropriate amounts of MT are dried in a hydrolysis tube, dissolved in 25- 50/d distilled formic acid, chilled, and mixed with 50100/d of cold performic acid (the final ratio of formic acid to performic acid is 1 : 2). After incubation for 3 hr on ice 25- 50 pl of water is added and the sample is dried in a Speed Vac concentrator (Savant, Hicksville, NY) or freeze-dried and hydrolyzed.
Acid Hydrolysis Oxidized or alkylated salt-free MT is hydrolyzed for 22 hr at 110 ° in vacuo or under argon in 6 M HC1. Recoveries of threonine and serine may be calculated by extrapolation to zero time after hydrolysis for 16, 40, and 72 hr.
Amino Acid Analysis of Acid Hydrolysate Normally, acid hydrolysates of MT are applied to a commercially available amino acid analyzer. With a Durrum D-500 amino acid analyzer the optimal amount of hydrolyzed protein for analysis is approximately 10 or 0.5 /tg using detection with ninhydrin or o-phthalaldehyde reagent, respectively. With analyzers from other manufacturers (model 420A, Applied Biosystems, Inc., Foster City, CA; or Amino Quant, Hewlett-Packard) accurate results are obtained using approximately 50 ng of sample.
[49] Amino Acid Sequence
Determination
By PETER E. HUNZIKER Introduction Free cysteine residues are usually not detectable as phenylthiohydantoin (PTH) derivatives obtained by Edman degradation. Because this amino acid represents up to 33% of all amino acid residues in metallothioneins (MTs), it must be chemically modified for the unambiguous determination of the primary structure of this protein. In addition to the variety of cysteine derivatives employed in peptide mappingm and sequence W. R. Berhnard, M. Va~k, and J. H. R. KAgi, Biochemistry 25, 1975 (1986).
METHODS IN ENZYMOLOGY, VOL. 205
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
422
CHEMICAL CHARACTERIZATION OF METALLOTHIONEINS
[49]
analysis, 3-7 good results were obtained in our laboratory using the S-methylated derivative of MT. 8 Complete primary structure of MT is achieved by N-terminal sequence analysis of the modified protein and by sequence analysis of peptides derived from a number of proteolytic digests. In this chapter the chemical and enzymatic fragmentations usually necessary for complete determination of the amino acid sequence of this protein are described. Material and Methods
Sequencing Equipment Gas-phase sequencer, model 470A/900A, equipped with an online PTH analyzer, model 120A (Applied Biosystems, Inc., Foster City, CA) Pulsed-liquid phase sequencer, model 477A, equipped with an online PTH analyzer, model 120A (Applied Biosystems, Inc.)
Chemicals Chemicals for sequence analysis: All are purchased from Applied Biosystems, Inc. Proteolytic enzymes ("sequencing grade"): All are supplied by Boehringer (Mannheim, Germany) and reconstituted according to instructions Trifluoroacetic acid (TFA) for high-performance liquid chromatography (HPLC) is obtained from Pierce (Rockford, IL) All other chemicals: All are of analytical or HPLC grade and are supplied by Fluka (Bucks, Switzerland) or by Merck Cysteine Modification. Purified apoMTs are modified by S-methylation prior to peptide mapping and sequence analysis, s,9 Acid Cleavage. This procedure is necessary only for vertebrate MTs containing an acetylated N terminus. Up to 1 nmol of S-methylated MT is 2 K. Mfinger and K. Lerch, Inorg. Chim. Acta 151, 11 (1988). 3 T. Kojima, C. Berger, and J. H. R. K~gi, in "Metallothionein" (J. H. R. K~gi and M. Nordberg, eds.), p. 153. Birlda~user, Basel, 1979. 4 K. M~nger, U. A. Germann, M. Beltramini, D. Niedermann, G. Baitella, J. H. R. K~gi, and K. Lerch, J. Biol. Chem. 260, 10032 (1985). 5 I.-Y. Huang, A. Yoshida, H. Tsunoo, and H. Nakajima, J. Biol. Chem. 252, 8217 (1977). 6 I.-Y. Huang, M. Kimura, A. Hata, H. Tsunoo, and A. Yoshida, J. Biochem. 89, 1839 (1981). 7D. R. Winge, K. B. Nielson, R. D. Zeikus, and W. R. Gray, J. Biol. Chem. 259, 11419 (1984). 8 p. E. Hunziker, this volume [45]. 9 p. E. Hunziker, this volume [53].
[49]
AMINO ACID SEQUENCE DETERMINATION
MT-2B
423
MT-2A
o~
6
