38
For more information, or to download product instructions,
Sample Preparation and Hydrolysis
The extraction and purification of proteins play an important
role in determining amino acid content. These methods are
based on one or more of their physical characteristics
(e.g., solubility, molecular size, charge, polarity and specific
covalent or noncovalent interactions). The techniques
commonly used to separate proteins and peptides include:
• Reverse-phase HPLC
• Polyacrylamide gel electrophoresis
• Gel filtration
• Ion exchange chromatography
• Affinity chromatography
Table 1 provides a more detailed list of methods for
fractionating peptide mixtures.
25
Hydrolysis
Most protein samples require some form of chemical
treatment before their component amino acids are suitable
for analysis. Protein and peptide samples must be hydro-
lyzed to free amino acids from peptide linkages. Acids
(usually HCI) are the most widely used agents for
hydrolyzing proteins.
A simplified hydrolysis procedure involves refluxing the
protein with excess HCI, then removing the excess acid in
vacuum.
26
The lyophilized protein then is suspended in
constant boiling 6 N HCI and introduced into the hydrolysis
tube. The sample is frozen by immersing the tube in dry ice
and acetone. Before sealing, the tube is evacuated to
avoid formation of cysteic acid, methionine sulfoxide and
chlorotyrosine.
27
This procedure minimizes de-
composition of reduced S-carboxymethylcysteine and
analyzes S-carboxymethylated proteins. Hydrolysis is
conducted at 110°C (with the temperature accurately
controlled) for 20-70 hours by Moore and Stein’s method.
28
After hydrolysis, residual HCI is removed in a rotary
evaporator. The residue is dissolved in water and brought to
the appropriate pH for addition to the analyzer column.
28
References
1. Braconnot, H. (1820).
Ann. Chem. Phys.
13
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Z. Anal. Chem.
22
, 366.
4. Rattenbury, J.M. (1981).
Amino Acid Analysis,
Ellow Horwood, publisher,
Chicester, England, Chapter 1.
5. Martin, A.J.P. and Synge, R.L.M. (1941).
Biochem. J.
35
,
1358.
6. Elsden, S.R. and Synge, R.L.M. (1944).
Proc. Biochem. Soc. IX.
7. Synge, R.L.M. (1944).
Biochem. J.
38
, 285.
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Biol.
14
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et al.
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(1966).
Anal. Biochem.
14
, 41.
12. Thomson, A.R. and Miles, B.J. (1964).
Nature
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, 483.
13. Robinson, G.W. (1975).
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3
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14. Benson, J.V. (1972).
Anal. Biochem.
50
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15. Benson, J.V. and Patterson, J.A. (1965).
Anal. Chem.
37
, 1108.
16. Bates, R.G. and Pinching, G.D. (1949).
J. Amer. Chem. Soc.
71
, 1274.
17. Jones, B.N. and Gilligan, J.P. (1983).
American Biotech. Lab.
December, 46-51.
18. Weidmeier, V.T.,
et al.
(1982).
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231
, 410-417
19. DeJong, C.,
et al.
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20. Lindroth, P. and Mopper, K. (1979).
Anal. Chem.
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, 1667-1674.
21. Jones, B.N.,
et al.
(1981).
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4
, 565-586.
22. Turnell, D.C. and Cooper, J.D.H. (1982).
CIin. Chem.
28
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23. Umagat, H.,
et al.
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25. AlIen, G. (1981). Laboratory Techniques in Biochemistry and Molecular Biology,
T.S. Work and R.H. Burdon, Eds., Elsevier/North-Holland, Biomedical Press.
26. Moore, S. and Stein, W.H. (1963).
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27. Eveleigh, J.W. and Winter, G.D. (1970). Protein Sequence Determination, S.B.
Needleman, Ed., Springer-Verlag, pp. 92-95.
28. Moore and Stein,
op. cit.
Table 1. Methods for the fractionation of peptide mixtures.
Technique
Properties of Peptide Molecules Exploited
Centrifugation
Solubility
Size exclusion chromatography
Size
Ion exchange chromatography
Charge, with some influence of polarity
Paper electrophoresis
Charge and size
Paper chromatography
Polarity
Thin layer electrophoresis
Charge and size
Thin layer chromatography
Polarity
Polyacrylamide gel electrophoresis
Charge and size
High-performance liquid chromatography (HPLC)
Polarity
Gas chromatography
Volatility of derivatives
Counter-current extraction
Polarity; sometimes specific interactions
Affinity chromatography
Specific interactions
Covalent chromatography or irreversible binding
Disulfide bond formation; reactivity of homoserine lactone
