Abstract
Use of insulin's intrinsic tyrosine absorption and fluorescence to monitor its interaction with the insulin receptor is limited because the spectral properties of the receptor tryptophan residues mask the spectral properties of the hormone tyrosine residues. We describe the synthesis of an insulin analog where A14 tyrosine is replaced by a tryptophan analog, 5-hydroxytryptophan. This insulin is spectrally enhanced since 5-hydroxytryptophan has an absorption band above 300 nm which is at lower energies than the absorption of tryptophan. Steady-state and time-resolved fluorescence parameters indicate that 5-hydroxytryptophan reports the same information about the environment of the A14 side chain as does the corresponding tryptophan-containing insulin. The synthetic hormone is a full agonist compared to porcine insulin, but has slightly reduced specific activity. Consequently, this spectrally enhanced insulin analog will be useful for hormone-receptor interaction studies since it can be observed by both absorption and fluorescence even in the presence of the tryptophan-containing receptor.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alcala, J. R., Gratton, E., and Prendergast, F. G. (1987).Biophys. J. 51, 925–936.
Badea, M. G., and Brand, L. (1979).Meth. Enzymol. 61, 378–425.
Barany, G., and Merrifield, R. B. (1980). InThe Peptides, Vol. 2 (Gross, E., and Meienhofer, J., eds.), Academic Press, New York, pp. 3–284.
Beechem, J. M., and Brand, L. (1985).Annu. Rev. Biochem. 54, 43–71.
Bevington, P. R. (1969).Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York.
Blundell, T. L., Dodson, G., Hodgkin, D. C., and Mercola, E. (1972).Adv. Protein Chem. 26, 270–402.
Burke, G. T., Hu, S. Q., Ohta, N., Schwartz, G. P., Zong, L., and Katsoyannis, P. G. (1990).Biochem. Biophys. Res. Commun. 173, 982–987.
Chance, R. E., Hoffman, J. A., Kroef, E. P., Johnson, M. G., Schmirer, E. W., Bromer, W. W., Ross, M. J., and Wetzel, R. (1981).Proc. Am. Pept. Symp. 7, 721–728.
Chen, R. F., Knutson, J. R., Ziffer, H., and Porter, D. (1991).Biochemistry 30, 5184–5195.
Chu, Y.-C., Burke, G. T., and Katsoyannis, P. G. (1992).J. Protein Chem. 11, 571–577.
Chu, Y.-C., Burke, G. T., Ross, J. B. A., and Katsoyannis, P. G. (1993).J. Protein Chem. 12, 499–505.
Eftink, M. R. (1991).Meth. Biochem. Anal. 35, 127–205.
Grinvald, A., and Steinberg, I. Z. (1974).Anal. Biochem. 59, 583–598.
Hasselbacher, C. A., Waxman, E., Galati, L. T., Contino, P. B., Ross, J. B. A., and Laws, W. R. (1991).J. Phys. Chem. 95, 2995–3005.
Hogue, C. W. V., Rasquinha, I., Szabo, A. G., and MacManus, J. P. (1992).FEBS Lett. 310, 269–272.
James, D. R., and Ware, W. R. (1985).Chem. Phys. Lett. 120, 450–454.
Joshi, S., Burke, G. T., and Katsoyannis, P. G. (1990).J. Protein Chem. 9, 235–246.
Katsoyannis, P. G. (1981). InStructural Studies on Molecules of Biological Interest (Dodson, G. G., Glusker, J. P., and Sayre, D., eds.), Oxford University Press, Oxford, pp. 454–486.
Katsoyannis, P. G., Tometsko, A., Zalut, C., Johnson, S., and Trakatellis, A. C. (1967).Biochemistry 6, 2635–2641.
Katsoyannis, P. G., Schwartz, G. P., Burke, G. T., Joshi, S., Rechler, M. M., and Tsang, L. Y.-H. (1987). InPeptides 1986 (Theodoropoulos, D., ed.), Walter de Gruyter, New York, pp. 43–60.
Kitagawa, K., Ogawa, H., Burke, G. T., Chanley, J. D., and Katsoyannis, P. G. (1984).Biochemistry 23, 4444–4448.
Kombo, D. C., Némethy, G., Gibson, K. D., Ross, J. B. A., Rackovsky, S., and Scheraga, H. A. (1995).J. Mol. Biol, submitted.
Laue, T. M., Senear, D. F., Eaton, S., and Ross, J. B. A. (1993).Biochemistry 32, 2469–2472.
Martinez, J., and Bodanszky, M. (1978).Int. J. Peptide Protein Res. 12, 277–283.
Rayner, D. M., and Szabo, A. G. (1978).Can. J. Chem. 56, 743–745.
Ross, J. B. A., Laws, W. R., Rousslang, K. W., and Wyssbrod, H. R. (1992a). InTopics in Fluorescence Spectroscopy, Volume 3: Biochemical Applications (Lakowicz, J. R., ed.), Plenum, New York, pp. 1–63.
Ross, J. B. A., Senear, D. F., Waxman, E., Kombo, B. B., Rusinova, E., Huang, Y. T., Laws, W. R., and Hasselbacher, C. A. (1992b).Proc. Natl. Acad. Sci. USA 89, 12023–12027.
Ross, J. B. A., Wyssbrod, H. R., Porter, R. A., Schwartz, G. P., Michaels, C. A., and Laws, W. R. (1992c).Biochemistry 31, 1585–1594.
Saito, I., Sugihama, H., Yamamoto, A., Muramatsu, S., and Matsuura, T. (1984).J. Am. Chem. Soc. 106, 4286–4287.
Shizuka, H., Serizawa, M., Shimo, T., Saito, I., and Matsuura, T. (1988).J. Am. Chem. Soc. 110, 1930–1934.
Szabo, A. G., and Rayner, D. M. (1980).J. Am. Chem. Soc. 102, 554–563.
Wagner, B. D., James, D. R., and Ware, W. R. (1987).Chem. Phys. Lett. 138, 181–184.
Waxman, E., Rusinova, E., Hasselbacher, C. A., Schwartz, G. P., Laws, W. R., and Ross, J. B. A. (1993).Anal. Biochem. 210, 425–428.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Laws, W.R., Schwartz, G.P., Rusinova, E. et al. 5-Hydroxytryptophan: An absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin. J Protein Chem 14, 225–232 (1995). https://doi.org/10.1007/BF01886763
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01886763