Summary
The use of hydrophobic-interaction chromatography (HIC) is proposed for the simultaneous determination of more than one thiol-protein after formation of the corresponding mercury mercaptides withp-hydroxymercuribenzoate (PHMB). The new chromatographic procedure, based on the HIC separation of the modified proteins from each other and from excess organomercury reagent has been successfully applied to the quantitative determination of phosphoglucose isomerase (PGI) and phosphoglucose mutase (PGM) in crude PGI powder, and of L-lactate dehydrogenase, PGM and aldolase in crude pyruvate kinase from rabbit muscle. The suitability of203Hg-labelled PHMB has been tested in the analysis of mixtures, which give barely distinguishable UV-peaks owing to the presence of other non-thiol components in the sample. For this purpose glyceraldehyde 3-phosphate dehydrogenase (GAPDHy) and PGIy from bakers yeast have been considered. Results obtained in experiments performed by both procedures are reported.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
P. C. Jocelyn (Editor), Biochemistry of the SH Group, Academic Press, New York, 1972.
M. Friedman (Editor), The Chemistry and Biochemistry of the Sulphydryl Group in Amino Acids, Peptides and Proteins, Pergamon Press, London, 1973.
G. Raspi, A. Lo Moro, M. Spinetti, G. Tesi, Chromatographia46, 471 (1997).
K. Shimada, K. Mitamura, J. Chromatogr. B,659, 227 (1994).
P. D. Boyer, J. Am. Chem. Soc.76, 4331 (1954).
H. Cerfontain, G. M. F. A. van Aken, J. Am. Chem. Soc.78, 2094 (1956).
R. Iyengar, I. A. Rose, Biochemistry20, 1223 (1981).
R. Jaenicke, S. Knof., Eur. J. Biochem.4, 157 (1968).
N. G. Pon, K. D. Schnacherz, M. N. Blackburn, G. C. Chatterjee, E. A. Noltmann, Biochemistry9, 1506 (1970).
W. J. Ray Jr, M. A. Hermodson, J. M. Puvathingal, W. C. Mahoney, J. Biol. Chem.258, 9166 (1983).
J. Milne, R. A. Cook, Biochemistry13, 4196 (1974).
T. D. Kempe, D. M. Gee, G. M. Hathaway, E. A. Noltmann, J. Biol. Chem.249, 4625 (1974).
A. D. Swenson, P. D. Boyer, J. Am. Chem. Soc.79, 2174 (1957).
O. P. Malhotra, D. K. Srivastava, A. M. Kayastha, Srinivasan, Indian J. Biochem. Biophys.30, 264 (1993).
G. C. Chatterjee, E. A. Noltmann, J. Biol. Chem.262, 3440 (1967).
V. Bocchini, M. R. Alioto, V. A. Najjar, Biochemistry6, 313 (1967).
G. Raspi, A. Lo. Moro, M. Spinetti, G. Tesi, Anal. Commun.34, 307 (1997).
V. J. Erwin, P. L. Pedersen, Anal. Biochem.25, 477 (1968).
Y. Ando, M. Steiner, Biochim. Biophys. Acta311, 38 (1972).
S. F. Velick, Symposium on the Mechanism of Enzyme Action, McCollum-Pratt Institute, Johns-Hopkins University, Contribution 70 491 (1954).
S. F. Velick, J. E. Hayes, J. Harting, J. Biol. Chem.203, 527 (1953).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Raspi, G., Moro, A.L., Spinetti, M. et al. p-Hydroxymercuribenzoate as thiol-protein modifier for simultaneous determination of glycolytic enzymes by hydrophobic-interaction chromatography. Chromatographia 49, 47–53 (1999). https://doi.org/10.1007/BF02467186
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02467186