Dityrosine-based substrates for the selective and sensitive assay of thermolysin

Chan-Jin Kim; Dong-Ik Lee; Chang-Ha Lee; Ik-Sung Ahn

Journal of Industrial and Engineering Chemistry, Vol.21, 248-253, January, 2015

DOI10.1016/j.jiec.2014.02.031 Export Citation

Dityrosine-based substrates for the selective and sensitive assay of thermolysin

Chan-Jin Kim, Dong-Ik Lee¹, Chang-Ha Lee , and Ik-Sung Ahn^†

Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea
¹Dityrosine Innovation Chemical (D. I. Chemical), B120C Engineering Research Center, Yonsei University, Seoul 120-749, South Korea

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Thermolysin family proteases are related to various diseases such as bacterial infections, cholera, gastritis and peptic ulcers, and gastric carcinoma. Hence, assay materials capable of the sensitive and specific assessment of thermolysin activity need to be developed for the detection of those diseases. In this study, simple peptides containing an unusual fluorescent amino acid, dityrosine, were tested for the assay of thermolysin. Various types of DBDY-(amino acid-INH)2 were synthesized from the conjugation of N,N0-diBoc-dityrosine (DBDY) with two molecules of amino acid and isoniazid (INH). Among these, DBDY-(Phe-INH)2 and DBDY-(Ile-INH)2 were found to be the most promising substrates for thermolysin assays. The hydrolysis reaction occurring between DBDY and Phe (or Ile) resulted in the release of Phe-INH and Ile-INH, and the fluorescence of DBDY was recovered. The reaction was found to follow Michaelis-Menten kinetics, and the KM and kcat/KM values were in the ranges of 1.91.3.95 mM and 2.43 × 10^(4)-6.03 × 104 M-1 s-1, respectively, which were comparable to other protease assay materials. Considering the simple preparations of DBDY-(Phe-INH)2 and DBDY-(Ile-INH)2, they are believed to be useful for the selective and sensitive assay of thermolysin.

Keywords:Dityrosine;Fluorescence;Thermolysin;Selective and sensitive assay

[References]

Polgar L, Mechanisms of Protease Action, CRC Press Inc., Florida, p. 183. (1990)
Adekoya OA, Sylte I, Chem. Biol. Drug Des., 73, 7 (2009)
Jin F, Matsushita O, Katayama S, Jin S, Matsushita C, Minami J, Okabe A, Infect. Immun., 64, 230 (1996)
Mariencheck WI, Alcorn JF, Palmer SM, Wright JR, Am. J. Respir. Cell Mol. Biol., 28, 528 (2003)
Hobden JA, DNA Cell Biol., 21, 391 (2002)
Naka A, Yamamoto K, Miwatani T, Honda T, FEMS Microbiol. Lett., 98, 197 (1992)
Schmidtchen A, Holst E, Tapper H, Bjorck L, Microb. Pathog., 34, 47 (2003)
Schmidtchen A, Wolff H, Rydengard V, Hansson C, Acta Derm.-Venereol., 83, 310 (2003)
Smith AW, Chahal B, French GL, Mol. Microbiol., 13, 153 (2006)
Waller CL, Oprea TI, Giolitti A, Marshall GR, J. Med. Chem., 36, 4152 (1993)
Waller CL, Marshall GR, J. Med. Chem., 36, 2390 (1993)
Marie-Claire C, Ruffet E, Antonczak S, Beaumont A, O’Donohue M, Roques BP, Fournie-Zaluski MC, Biochemistry, 36, 13938 (1997)
Spungin-Bialik A, Ben-Meir D, Fudim E, Carmeli S, Blumberg S, Febs Lett., 380, 79 (1996)
Marguerre AK, Kramer R, Bioorg. Med. Chem. Lett., 19, 5757 (2009)
Ghale G, Ramalingam V, Urbach AR, Nau WM, J. Am. Chem. Soc., 133(19), 7528 (2011)
Marme N, Knemeyer JP, Wolfrum J, Sauer M, Angew. Chem. Int. Ed., 43, 3798 (2004)
Weimer S, Oertel K, Fuchsbauer HL, Anal. Biochem., 352, 110 (2006)
Giulivi C, Traaseth N, Davies K, Amino Acids, 25, 227 (2003)
Giulivi C, Davies KJA, Methods Enzymol., 233, 363 (1994)
Malencik D, Anderson S, Amino Acids, 25, 233 (2003)
Gross AJ, Sizer IW, J. Biol. Chem., 234, 1611 (1959)
Beynon R, Bond J, Proteolytic Enzymes, second ed., Oxford University Press, New York, pp. 295-316. (2001)
Kim CJ, Lee DI, Lee CH, Ahn IS, Anal. Chim. Acta, 723, 101 (2012)
Schechter I, Berger A, Biochem. Biophys. Res. Commun., 27, 157 (1967)
Lee DI, Choi JY, Kim CJ, Ahn IS, Process Biochem., 46, 142 (2011)
Kim CJ, Lee DI, Zhang D, Lee CH, Ahn IS, Anal. Biochem., 435, 166 (2013)
Shuler ML, Kargi F, Bioprocess Engineering Basic Concepts, second ed., Prentice-Hall Inc., New Jersey, pp. 57-104. (2001)

[Referenced By]

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[1] Polgar L, Mechanisms of Protease Action, CRC Press Inc., Florida, p. 183. (1990)

[2] Adekoya OA, Sylte I, Chem. Biol. Drug Des., 73, 7 (2009)

[3] Jin F, Matsushita O, Katayama S, Jin S, Matsushita C, Minami J, Okabe A, Infect. Immun., 64, 230 (1996)

[4] Mariencheck WI, Alcorn JF, Palmer SM, Wright JR, Am. J. Respir. Cell Mol. Biol., 28, 528 (2003)

[5] Hobden JA, DNA Cell Biol., 21, 391 (2002)

[6] Naka A, Yamamoto K, Miwatani T, Honda T, FEMS Microbiol. Lett., 98, 197 (1992)

[7] Schmidtchen A, Holst E, Tapper H, Bjorck L, Microb. Pathog., 34, 47 (2003)

[8] Schmidtchen A, Wolff H, Rydengard V, Hansson C, Acta Derm.-Venereol., 83, 310 (2003)

[9] Smith AW, Chahal B, French GL, Mol. Microbiol., 13, 153 (2006)

[10] Waller CL, Oprea TI, Giolitti A, Marshall GR, J. Med. Chem., 36, 4152 (1993)

[11] Waller CL, Marshall GR, J. Med. Chem., 36, 2390 (1993)

[12] Marie-Claire C, Ruffet E, Antonczak S, Beaumont A, O’Donohue M, Roques BP, Fournie-Zaluski MC, Biochemistry, 36, 13938 (1997)

[13] Spungin-Bialik A, Ben-Meir D, Fudim E, Carmeli S, Blumberg S, Febs Lett., 380, 79 (1996)

[14] Marguerre AK, Kramer R, Bioorg. Med. Chem. Lett., 19, 5757 (2009)

[15] Ghale G, Ramalingam V, Urbach AR, Nau WM, J. Am. Chem. Soc., 133(19), 7528 (2011)

[16] Marme N, Knemeyer JP, Wolfrum J, Sauer M, Angew. Chem. Int. Ed., 43, 3798 (2004)

[17] Weimer S, Oertel K, Fuchsbauer HL, Anal. Biochem., 352, 110 (2006)

[18] Giulivi C, Traaseth N, Davies K, Amino Acids, 25, 227 (2003)

[19] Giulivi C, Davies KJA, Methods Enzymol., 233, 363 (1994)

[20] Malencik D, Anderson S, Amino Acids, 25, 233 (2003)

[21] Gross AJ, Sizer IW, J. Biol. Chem., 234, 1611 (1959)

[22] Beynon R, Bond J, Proteolytic Enzymes, second ed., Oxford University Press, New York, pp. 295-316. (2001)

[23] Kim CJ, Lee DI, Lee CH, Ahn IS, Anal. Chim. Acta, 723, 101 (2012)

[24] Schechter I, Berger A, Biochem. Biophys. Res. Commun., 27, 157 (1967)

[25] Lee DI, Choi JY, Kim CJ, Ahn IS, Process Biochem., 46, 142 (2011)

[26] Kim CJ, Lee DI, Zhang D, Lee CH, Ahn IS, Anal. Biochem., 435, 166 (2013)

[27] Shuler ML, Kargi F, Bioprocess Engineering Basic Concepts, second ed., Prentice-Hall Inc., New Jersey, pp. 57-104. (2001)