Summary
Formation and dissociation of the benzamidine: β-trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine: β-trypsin complex minus the free proteinase) is 1.75 mM−1 cm−1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the β-trypsin: BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 ± 0.5 °C. Under all the experimental conditions the β-trypsin: BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine: β-trypsin:BPTI adduct (7). The difference between the kinetic processes of β-trypsin: BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S1 subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.
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Abbreviations
- BPTI:
-
bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor)
- pNGB:
-
p-nitrophenyl-p-guanidinobenzoate
- NaDodSO4 :
-
sodium dodecyl sulfate
References
Bernhard SA, Gutfreund H: Proc Natl Acad Sci USA 53: 1238–1243, 1965.
Glazer AN: Proc Natl Acad Sci USA 54: 171–176, 1965.
Bernhard SA, Lee BF, Tashjian ZH: J Mol Biol 18: 405–420, 1966.
Himoe A, Brandt KG, Hess GP: J Biol Chem 242: 3963–3972, 1967.
Himoe A, Brandt KG, De Sa RJ, Hess GP: J Biol Chem 244: 3483–3493, 1969.
Fersht AR, Requena Y: J Mol Biol 60: 279–290, 1971.
Antonini E, Ascenzi P, Menegatti E, Guarneri M: Biopolymers 22: 363–375, 1983.
Antonini E, Ascenzi P, Bolognesi M, Guarneri M, Menegatti E: Symposia Biologica Hungarica 25: 325–338, 1984.
Antonini E, Ascenzi P, Bolognesi M, Menegatti E, Guarneri M: J Biol Chem 258: 4676–4678, 1983.
Menegatti E, Guarneri M, Bolognesi M, Ascenzi P, Antonini F: Il Farmaco 38: 978–997, 1983.
Quast U, Engel J, Heumann H, Krause G, Steffen E: Biochemistry 17: 2512–2520, 1974.
Guillain F, Thusius D: J Am Chem Soc 92: 5534–5536, 1970.
Quast U, Engel J, Steffen E, Tschesche H, Kupfer S: Biochemistry 17: 1675–1682, 1978.
Goto S, Hess GP: J Biochem (Tokio) 85: 961–965, 1979.
Antonini E, Ascenzi P, Menegatti E, Bortolotti F, Guarneri M: Biochemistry 21: 2577–2482, 1982.
Ascenzi P, Menegatti E, Guarneri M, Bortolotti F, Antonini E: Biochemistry 21: 2483–2490, 1982.
East EJ, Trowbridge CG: Arch Biochem Biophys 125: 334–343, 1968.
Mares-Guia M, Shaw E: J Biol Chem 240: 1579–1585, 1965.
Bode W, Schwager P: J Mol Biol 98: 693–717, 1975.
Bode W, Chen Z, Bartels K, Kutzback C, Schmidt-Kastner G, Bartunik H: J Mol Biol 164: 237–282, 1983.
Schroeder T Jr, Shaw E: J Biol Chem 243: 2943–2949, 1968.
Luthy JA, Praissman M, Finkenstadt WR, Laskowski M Jr: J Biol Chem 248: 1760–1771, 1973.
Hruska JF, Law JH, Kézdy FJ: Biochem Biophys Res Commun 36: 272–277, 1969.
Sach E, Thély M, Choay H: Compt Rend Acad Sci Paris 260: 3491–3493, 1965.
Weber K, Pringle JR, Osborn M: Methods Enzymol 26: 2–27, 1972.
Vincent JP, Lazdunski M: FEBS Lett 63: 240–244, 1976.
Antonini E, Ascenzi P, Bolognesi M, Gatti G, Guarneri M, Menegatti E: J Mol Biol 165: 543–558, 1983.
Bode W: J Mol Biol 127: 357–374, 1979.
Menegatti E, Guarneri M, Ferroni R, Bolognesi M, Ascenzi P, Antonini E: FEBS Lett 141: 33–36, 1982.
Evans SA, Olson ST, Shore JD: J Biol Chem 257: 3014–3017, 1982.
Atkins GL, Nimmo JA: Biochem J 135: 779–784, 1973.
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Ascenzi, P., Bolognesi, M., Guarneri, M. et al. Benzamidine as a spectroscopic probe for the primary specificity subsite of trypsin-like serine proteinases. Mol Cell Biochem 64, 139–144 (1984). https://doi.org/10.1007/BF00224770
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DOI: https://doi.org/10.1007/BF00224770