Kenneth W. Rousslang

Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides

The fluorescence and phosphorescence of proteins and polypeptides is the sum of the contributions from the three aromatic amino acids tryptophan, tyrosine, and phenylalanine. The work on protein and polypeptide luminescence prior to 1971 has been reviewed in detail by Longworth. Another fine account of the early work, emphasizing tryptophan and tyrosine, is the monograph by Konev. An excellent review on tyrosine fluorescence in proteins and model peptides, for the period up to 1975, is given by Cowgill. In 1984, Creed reviewed the photophysics and photochemistry of tyrosine and its simple derivatives, including a thorough coverage of steady-state fluorescence and a brief discussion of triplet-state properties, but did not include any work on proteins or polypeptides. The first quantitative studies of the excited-state properties of the three aromatic amino acids were carried out in the 1950s. The low-temperature phosphorescence of the aromatic amino acids was initially observed by Debye and Edwards in 1952, and phosphorescence emission spectra were reported by Steele and Szent-Gyorgyi in 1957. In 1953, Weber postulated that the fluorescence of the aromatic amino acids should occur in the near-ultraviolet region of the electromagnetic spectrum. In 1956, independently and almost simultaneously, Duggan and Udenfriend and Shore and Pardee reported the results of their investigations of protein fluorescence. At the same time,

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins.

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.

Triplet state decay and spin-lattice relaxation rate constants in tyrosinate and tryptophan

Abstract The decay rates and spin-lattice relaxation rates are presented for the lowest excited triplet state in tryptophan and in tyrosinate in zero-field at 1.34 K.

[12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage.

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.

Triplet-triplet energy transfer in the tryptophyl-tyrosinate dipeptide☆

Abstract Direct evidence for intermolecular triplet-triplet energy transfer in the dipeptide tryptophyl-tyrosine is presented. The methods of steady state ODMR and transient ODMR yield rates for the triplet-triplet energy transfer process of order 10 s −1 .

PHOSPHORESCENCE MAXIMA AND TRIPLET STATE LIFETIMES OF NAD + AND ε‐NAD+ IN TERNARY COMPLEXES WITH HORSE LIVER ALCOHOL DEHYDROGENASE

Abstract This paper describes the phosphorescence emission and decay times of NAD and its fluorescent etheno derivative, ε‐NALV+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the ε‐NAD+, triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme‐enzyme interaction. The decays of NAD+ and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in ε‐NAD4 than they are in ε‐AMP. Whereas the decay of ε‐AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of e‐NAD+ to a double exponential. Ternary complexes with NAD+ excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the e‐NAD+ ternary complex is provided at 313 nm. where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of ε‐NAD+ in solution. Our results demonstrate that NAD+ and ε‐NAD+ quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0–0 vibronic band assigned to Trp‐15 (blue‐edge), we calculate a 21% decrease in the phosphorescence associated with Trp‐314 at stoichiometric saturation of the coenzyme binding sites with NAD‐ in the ternary complex. When the active sites are saturated with εS‐NAD+, the relative phosphorescence due to Trp‐314 decreases by 63%. Since ε‐NAD+ can be selectively excited in ternary complexes with ADH, it is a more useful probe than NAD+ for investigating energy transfer in these complexes.

Time-Resolved Phosphorescence of Tyrosine, Tyrosine Analogs, and Tyrosyl Residues in Oxytocin and Small Peptides

We present the time-resolved phosphorescence of oxytocin, two oxytocin derivatives, vasopressin and a series of compounds that serve as models for free tyrosine. One of the oxytocin derivatives, desaminodicarbaoxytocin, has the disulfide bridge replaced by an ethylene bridge, and lacks the N-terminus. Similar to the reported fluorescence decays of tyrosine in these peptides, the phosphorescence decays generally are not single exponentials, but can be fit as biexponentials. The decay times for the oxytocin peptides are shorter than for desaminodicarbaoxytocin or the model compounds, and this we attribute to enhanced spin-orbit coupling due to the presence of sulfur. We measured the phosphorescence decay of the model cyclic pentapeptide that contains tyrosine and compared it to that observed for the same cyclic pentapeptide in which tyrosine is replaced by tryptophan. We also report the phosphorescence of 2-tryptophan-oxytocin, and deamino-2-tryptophan-oxytocin in which biexponential phosphorescence decay is also observed.

Steady-state and time-resolved phosphorescence of wild-type and modified bacteriophage λcI repressors.

We have measured the steady-state phosphorescence and decay times of wild-type λcI repressor and compared it with that of a modified λcI repressor in which > 95% of the tryptophans were replaced with 5-hydroxy-l-tryptophan (5-OHTrp). The wild-type and 5-OHTrp-λcI repressors are spectroscopically distinct such that we can selectively excite the 5-OHTrp-λcI even in the presence of a 15-fold molar excess ofN-acetyltryptophanamide (NATrpA). The phosphorescence band of wild-type λcI is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan. Similarly, the phosphorescence of 5-OHTrp-λcI repressor is red-shifted relative to the model, 5-OHTrp, showing that according to the phosphorescence, the modified repressor is structurally indistinguishable from the native repressor. While the phosphorescence decay of both NATrpA and 5-OHTrp are single exponentials, the decay of both wild-type and 5-OHTrp-λcI repressors is complex, requiring three decay components whose fractional contributions to the phosphorescence are the same for both repressors. Because the 5-OHTrp phosphorescence can be excited at wavelengths outside the absorbance range of tryptophan and DNA, a protein spectrally enhanced with this emitter will aid the investigations of protein-protein or protein-DNA interactions.

Steady-state and time-resolved phosphorescence of 5-hydroxy-L-tryptophan lambda cI repressor bound to DNA

The spectral overlap of tryptophan containing proteins and DNA has limited the use of luminescence methods to investigate protein-nucleic acid interactions. However, the steady-state and time-dependent phosphorescence of wild-type and 5-hydroxy-L-tryptophan (5-O1-ITrp) X ci repressor are spectroscopically distinct such that we can selectively excite the 5-OHTrp-?. ci in the presence of DNA, or even in the presence of a 15-fold molar excess of N-acetyl-tryptophanamide (NATrpA). The phosphorescence of wild-type X ci is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan, and the phosphorescence of the spectrally enhanced protein (SEP), 5-OHTrp-X ci repressor is also red shifted relative to the model, 5-OHTrp, providing spectroscopic evidence that the modified repressor is structurally equivalent to the native repressor. Although the phosphorescence decays of NATrpA and 5-OHTrp are simple exponentials, the decay of either wild-type or 5-OHTrp-X ci repressors requires three exponentials whose fractional contributions to the phosphorescence are similar. Since the 5-OFITrp phosphorescence can be excited without interference from tryptophan or DNA, we measured the phosphorescence of the SEP/DNA complex. Th emission characteristics of SEP alone and the SEP/DNA complex are indistinguishable, showing that during binding, the C-terminal domain of the protein, believed to be involved in protein dimer stabilization, is structurally conserved in the vicinity of the three modified trytptophans.

Time-resolved phosphorescence of proteins and polypeptides

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.