Time-resolved phosphorescence of proteins and polypeptides

Kenneth W. Rousslang; Steve K. Buratto; D. Haynes; P. Heath; D. Holloway; John C. Hulteen; Lisa Dick; K. Stevenson; C. O. Harding; J. B. Alexander Ross

doi:10.1117/12.17735

1 May 1990 Time-resolved phosphorescence of proteins and polypeptides

Kenneth W. Rousslang, Steve K. Buratto, D. Haynes, P. Heath, D. Holloway, John C. Hulteen, Lisa Dick, K. Stevenson, C. O. Harding, J. B. Alexander Ross

Author Affiliations +

Proceedings Volume 1204, Time-Resolved Laser Spectroscopy in Biochemistry II; (1990) https://doi.org/10.1117/12.17735
Event: OE/LASE '90, 1990, Los Angeles, CA, United States

Abstract

This paper describes the use of luminescent coenzymes and substrates in the study of two proteins, horse liver alcohol dehydrogenase (ADH) and sex steroid-binding protein (SBP). We report the phosphorescence emission and lifetimes of NAD and e-NAD in pyrazole ternary complexes with ADH. Whereas the decay of NAD is adequately described as monoexponential with a lifetime of 2.4 s, the decay of e-NAD obeys a double exponential decay law with time constants of 0.4 and 0.15 sec. Ternary complexes with NAD have the same decay kinetics as ADH by itself. However, in ternary complexes with f-NAD, it is possible to selectively excite and detect the coenzyme phosphorescence. We show that f-NAD is a more useful probe than NAD for protein structure in ADH. We also measured the triplet emission and decay lifetimes of dihydroequilenin, an equine steroid bound by SBP. We find that the phosphorescence spectra and lifetimes are pH dependent, with the protonated species dominating emission below pH 10.0, and the deprotonated form dominating at pH 10.0 and above. The acidic and basic species can be selectively excited, and the emission at pH near the pKa is characteristic of the equilibrium ground state populations. Since hydrogen bonding is implicated in SBP-steroid complexes, dihydroequilenin is a potential phosphorescent probe for the binding interaction.

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Kenneth W. Rousslang, Steve K. Buratto, D. Haynes, P. Heath, D. Holloway, John C. Hulteen, Lisa Dick, K. Stevenson, C. O. Harding, and J. B. Alexander Ross "Time-resolved phosphorescence of proteins and polypeptides", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); https://doi.org/10.1117/12.17735

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KEYWORDS

Phosphorescence

Proteins

Biochemistry

Laser spectroscopy

Absorption

Spectroscopy

Amplifiers

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