142
Assay for studying kinetic properties of dipeptidases / V. Pandya et al. / Anal. Biochem. 418 (2011) 134–142
[11] J. Shi, J. Dertouzos, A. Gafni, D. Steel, Application of single-molecule
spectroscopy in studying enzyme kinetics and mechanism, Methods
Enzymol. 450 (2008) 129–157.
[12] N. Day, J.W. Keillor, A continuous spectrophotometric linked enzyme assay for
transglutaminase activity, Anal. Biochem. 274 (1999) 141–144.
[13] B.M. Dunn, M. Jimenez, B.F. Parten, M.J. Valler, C.E. Rolph, J. Kay, A systematic
series of synthetic chromophoric substrates for aspartic proteinases, Biochem.
J. 237 (1986) 899–906.
[14] L. Josefsson, T. Lindberg, Intestinal dipeptidases: I. Spectrophotometric
determination and characterization of dipeptidase activity in pig intestinal
mucosa, Biochim. Biophys. Acta 105 (1965) 149–161.
[15] R.C. Thompson, E.R. Blout, Dependence of the kinetic parameters for elastase-
catalyzed amide hydrolysis on the length of peptide substrates, Biochemistry
12 (1973) 57–65.
studying kinetics of hydrolysis of small peptides by peptidases, the
development of a simple and rapid MS method for determination
of kinetic parameters for peptidases is of great interest because
many peptidases are drug targets. In this study, we used ESI–MS
to characterize kinetic parameters of three peptidases and also
quantitated carnosine levels in vivo. Carnosine is an important bio-
active peptide, and development of a rapid quantitative assay is
useful for detecting the amount of carnosine in clinical samples.
Compared with previously described fluorescence and HPLC-based
assays that have sensitivity and time issues, the MS system pro-
vides a rapid, robust, and accurate method that can be further ex-
tended for studying kinetic properties of other dipeptidases. The
reported HPLC-based system needs to resolve the peaks of sub-
strates and products, which is sometimes difficult, and also needs
a time-intensive gradient run (50–60 min per HPLC run vs. 5–
6 min per run with this assay). The developed ESI–MS analysis
technique displays linearity over a wide range of analyte concen-
trations, and the intensity normalization procedure followed can
be easily adopted for any peptidase assay. Considering that a large
number of assays are needed to study the kinetic mechanism of
peptidases, the ESI–MS technique is more suitable in terms of both
accuracy and time, and nearly 15 times more assays can be per-
formed in a day without sacrificing reliability of reactant detection
and quantification. The HPLC and spectroscopy methods are diffi-
cult to implement for biological samples. Time-dependent quanti-
tation of carnosine in CHO cells using the ESI–MS method indicates
that ESI–MS can be used in a wide range of clinical and biological
studies for discerning the role of dipeptides in different biological
processes.
[16] D. Nardiello, T.R. Cataldi, Determination of carnosine in feed and meat by high-
performance anion-exchange chromatography with integrated pulsed
amperometric detection, J. Chromatogr. A 1035 (2004) 285–289.
[17] H. Otani, N. Okumura, A. Hashida-Okumura, K. Nagai, Identification and
characterization of
a mouse dipeptidase that hydrolyzes L-carnosine, J.
Biochem. 137 (2005) 167–175.
[18] M. Shikita, J.W. Fahey, T.R. Golden, W.D. Holtzclaw, P. Talalay, An unusual case
of ‘‘uncompetitive activation’’ by ascorbic acid: purification and kinetic
properties of a myrosinase from Raphanus sativus seedlings, Biochem. J. 341
(1999) 725–732.
[19] M.H. Elliott, D.S. Smith, C.E. Parker, C. Borchers, Current trends in quantitative
proteomics, J. Mass Spectrom. 44 (2009) 1637–1660.
[20] A. Liesener, U. Karst, Monitoring enzymatic conversions by mass
spectrometry: a critical review, Anal. Bioanal. Chem. 382 (2005) 1451–1464.
[21] D. Ganguli, C. Kumar, A.K. Bachhawat, The alternative pathway of glutathione
degradation is mediated by a novel protein complex involving three new genes
in Saccharomyces cerevisiae, Genetics 175 (2007) 1137–1151.
[22] J.F. Lenney, Separation and characterization of two carnosine-splitting
cytosolic dipeptidases from hog kidney (carnosinase and non-specific
dipeptidase), Biol. Chem. Hoppe Seyler 371 (1990) 433–440.
[23] M. Teufel, V. Saudek, J.P. Ledig, A. Bernhardt, S. Boularand, A. Carreau, N.J.
Cairns, C. Carter, D.J. Cowley, D. Duverger, A.J. Ganzhorn, C. Guenet, B.
Heintzelmann, V. Laucher, C. Sauvage, T. Smirnova, Sequence identification
and characterization of human carnosinase and a closely related non-specific
dipeptidase, J. Biol. Chem. 278 (2003) 6521–6531.
[24] H. Kaur, C. Kumar, C. Junot, M.B. Toledano, A.K. Bachhawat, Dug1p is a Cys-Gly
peptidase of the c-glutamyl cycle of Saccharomyces cerevisiae and represents a
Acknowledgments
novel family of Cys-Gly peptidases, J. Biol. Chem. 284 (2009) 14493–14502.
[25] J.F. Lenney, R.P. George, A.M. Weiss, C.M. Kucera, P.W. Chan, G.S. Rinzler,
Human serum carnosinase: characterization, distinction from cellular
carnosinase, and activation by cadmium, Clin. Chim. Acta 123 (1982) 221–231.
[26] H. Kaur, M. Datt, M.K. Ekka, M. Mittal, A.K. Singh, S. Kumaran, Cys-Gly specific
dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and
tetra-peptides: Peptide–protein interaction, homology modeling, and activity
studies reveal a latent promiscuity in substrate recognition, Biochimie 93
(2011) 175–186.
We thank Sekhar Majumdar for the support provided for cell
culture experiments and Sharanjit Kaur for technical help in MS
experiments. We thank Samir K. Nath for technical help in HPLC
experiments. V.P., M.K.E., and R.K.D. are Council of Scientific and
Industrial Research (CSIR) senior research fellows. This work was
supported by CSIR (India).
[27] J.F. Lenney, S.C. Peppers, C.M. Kucera-Orallo, R.P. George, Characterization of
human tissue carnosinase, Biochem. J. 228 (1985) 653–660.
[28] D. Su, K. Bi, C. Zhou, Y. Song, B. Wei, L. Geng, W. Liu, X. Chen, Enantioselective
separation and determination of carnosine in rat plasma by fluorescence LC for
stereoselective pharmacokinetic studies, Chromatographia 71 (2010) 603–
608.
[29] E.V. Berdyshev, I.A. Gorshkova, J.G. Garcia, V. Natarajan, W.C. Hubbard,
Quantitative analysis of sphingoid base-1-phosphates as bisacetylated
derivatives by liquid chromatography–tandem mass spectrometry, Anal.
Biochem. 339 (2005) 129–136.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
[30] D. Bungert, E. Heinzle, A. Tholey, Quantitative matrix-assisted laser
desorption/ionization mass spectrometry for the determination of enzyme
activities, Anal. Biochem. 326 (2004) 167–175.
[31] E. Stokvis, H. Rosing, J.H. Beijnen, Stable isotopically labeled internal standards
in quantitative bioanalysis using liquid chromatography/mass spectrometry:
necessity or not?, Rapid Commun Mass Spectrom. 19 (2005) 401–407.
[32] P. Mineo, D. Vitalini, D. La Mendola, E. Rizzarelli, E. Scamporrino, G. Vecchio,
References
[1] M. Babizhayev, Y. Courbebaisse, J.-F. Nicolay, Y. Semiletov, Design and
biological activity of imidazole-containing peptidomimetics with a broad-
spectrum antioxidant activity, Int. J. Pept. Res. Ther. 5 (1998) 163–169.
[2] R. Kohen, Y. Yamamoto, K.C. Cundy, B.N. Ames, Antioxidant activity of
carnosine, homocarnosine, and anserine present in muscle and brain, Proc.
Natl. Acad. Sci. USA 85 (1988) 3175–3179.
[3] S.H. Snyder, Brain peptides as neurotransmitters, Science 209 (1980) 976–983.
[4] W.T. Lowther, B.W. Matthews, Metalloaminopeptidases: common functional
themes in disparate structural surroundings, Chem. Rev. 102 (2002) 4581–
4608.
[5] A. Taylor, Aminopeptidases: structure and function, FASEB J. 7 (1993) 290–298.
[6] Y. Sato, Role of aminopeptidase in angiogenesis, Biol. Pharm. Bull. 27 (2004)
772–776.
[7] N.D. Rawlings, A.J. Barrett, MEROPS: the peptidase database, Nucleic Acids Res.
27 (1999) 325–331.
Electrospray mass spectrometric studies of L-carnosine (b-alanyl-L-histidine)
complexes with copper(II) or zinc ions in aqueous solution, Rapid Commun.
Mass Spectrom. 16 (2002) 722–729.
[33] A. Pegova, H. Abe, A. Boldyrev, Hydrolysis of carnosine and related compounds
by mammalian carnosinases, Comp. Biochem. Physiol. B 127 (2000) 443–446.
[34] N. Pi, J.I. Armstrong, C.R. Bertozzi, J.A. Leary, Kinetic analysis of NodST
sulfotransferase using an electrospray ionization mass spectrometry assay,
Biochemistry 41 (2002) 13283–13288.
[35] H. Gao, J.A. Leary, Kinetic measurements of phosphoglucomutase by direct
analysis of glucose-1-phosphate and glucose-6-phosphate using ion/molecule
reactions and Fourier transform ion cyclotron resonance mass spectrometry,
Anal. Biochem. 329 (2004) 269–275.
[36] Q. Chen, B. Zhang, L.M. Hicks, S. Wang, J.M. Jez, A liquid chromatography–
tandem mass spectrometry-based assay for indole-3-acetic acid-amido
synthetase, Anal. Biochem. 390 (2009) 149–154.
[37] C.J. Zea, N.L. Pohl, Kinetic and substrate binding analysis of phosphorylase b via
electrospray ionization mass spectrometry: a model for chemical proteomics
of sugar phosphorylases, Anal. Biochem. 327 (2004) 107–113.
[8] A.J. Turner, Exploring the structure and function of zinc metallopeptidases: old
enzymes and new discoveries, Biochem. Soc. Trans. 31 (2003) 723–727.
[9] S. Sauerhofer, G. Yuan, G.S. Braun, M. Deinzer, M. Neumaier, N. Gretz, J. Floege,
W. Kriz, F. van der Woude, M.J. Moeller,
L-Carnosine, a substrate of
carnosinase-1, influences glucose metabolism, Diabetes 56 (2007) 2425–2432.
[10] G. Vistoli, A. Pedretti, M. Cattaneo, G. Aldini, B. Testa, Homology modeling of
human serum carnosinase, a potential medicinal target, and MD simulations of
its allosteric activation by citrate, J. Med. Chem. 49 (2006) 3269–3277.