Characterization of the tryptophan residues of Escherechia coli alkaline phosphatase by phosphorescence and optically detected magnetic resonance spectroscopy.

The phosphorescence and zero field optically detected magnetic resonance (ODMR) of the tryptophan (Trp) residues of alkaline phosphatase from Escherechia coli are examined. Each Trp is resolved optically and identified with the aid of the W220Y mutant and the terbium complex of the apoenzyme. Trp(109), known from earlier work to be the source of room-temperature phosphorescence (RTP), emits a highly resolved low-temperature phosphorescence (LTP) spectrum and has the narrowest ODMR bands observed thus far from any protein site, revealing a uniquely homogeneous local environment. The decay kinetics of Trp(109) at 1.2 K reveals that the major triplet population (70%) undergoes inefficient crystallike spin-lattice relaxation by direct interaction with lattice phonons, the remainder being relaxed efficiently by local disorder modes. The latter population is smaller than is typical for protein sites, suggesting an unusual degree of local rigidity and order consistent with the long-lived RTP. Trp(220) emits a broader LTP spectrum originating to the blue of Trp(109). It has typically broad ODMR bands consistent with local heterogeneity. The LTP of Trp(268) has an ill-defined origin blue shifted relative to Trp(220) and ODMR frequencies consistent with a greater degree of solvent exposure. Trp(268) has noticeable dispersion of its decay kinetics, consistent with quenching at the triplet level by a nearby disulfide residue.

Determination of relative triplet sublevel populating rates during optical pumping using ODMR.

A method is introduced, based on optical detection of triplet state magnetic resonance (ODMR), to determine the relative populating rates of photoexcited triplet state sublevels during optical pumping. Phosphorescence transients induced by microwave rapid passage during optical pumping are analyzed globally utilizing kinetic parameters obtained from separate microwave-induced delayed phosphorescence measurements to obtain relative sublevel populating rates. Results are unaffected by phosphorescence from triplet populations that do not yield an ODMR response. The method is applied to the triplet state of the indole chromophore in various environments to reveal the effects of local interactions on the pattern of intersystem crossing. Enhanced spin--orbit coupling effects are attributed to interactions that reduce the planar symmetry of the indole chromophore.

Phosphorescence and optically detected magnetic resonance of HIV-1 nucleocapsid protein complexes with stem-loop sequences of the genomic Psi-recognition element.

The binding of NCp7, the nucleocapsid protein of human immunodeficiency virus type 1, to oligonucleotide stem--loop (SL) sequences of the genomic Psi-recognition element has been studied using fluorescence, phosphorescence, and optically detected magnetic resonance (ODMR). RNA SL2, SL3, and SL4 constructs bind with higher affinity than the corresponding DNAs. G to I substitutions in the SL3 DNA loop sequence lead to reduced binding affinity and significant changes in the triplet state properties of Trp37 of NCp7, implicating these bases in contacts with aromatic amino acid residues of the zinc finger domains of NCp7, in agreement with the NMR structure of the 1:1 complex of NCp7 and SL3 RNA [DeGuzman, R. N., Wu, Z. R., Stalling, C. C., Pappaladro, L., Borer, P. N., and Summers, M. F. (1998) Science 279, 384-388]. The NCp7 to SL binding stoichiometry is 2:1 for intact SL sequences but is reduced to 1:1 for SL variants with an abasic or hydrocarbon loop. It is proposed that Delta D/Delta E(0,0), where Delta D is the change in the zero-field splitting D parameter and Delta E(0,0) is the shift of the tryptophan phosphorescence origin, provides a measure of aromatic stacking interactions with nucleic acid bases. Values on the order of 10(-5) indicate significant stacking interactions, while values closer to 10(-6) result from interactions not involving aromatic stacking. Binding of NCp7 to oligonucleotide substrates produces shortened Trp37 triplet state lifetimes by enhancement of k(x) and an increase of the relative value of P(x), the intersystem crossing rate to the T(x) sublevel. These effects are attributed to a reduction in the degree of electronic symmetry of Trp37 in the complexes. Guanine and adenine triplet states produced by optical pumping of SL3 DNA are characterized. We find, as with tryptophan, that D < 3E.

Effect of nucleic acid binding on the triplet state properties of tetrapeptides containing tryptophan and 6-methyltryptophan: a study by phosphorescence and ODMR spectroscopy.

Complexes of four peptides [KWGK, KGWK, K(6MeW)GK, KG(6MeW)K] with the nucleic acids [poly(A), poly(C), poly(U), poly(I), and rG(8)] have been investigated by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The intrinsic spectroscopic probes used in these studies are tryptophan (W) and 6-methyltryptophan (6MeW). Binding to the nucleic acids results in a red-shift of the phosphorescence 0,0-band (delta E(0,0)) of the aromatic residue as well as a reduction of its zero-field splitting parameter (delta D). Results are compared with earlier studies of the HIV-1 nucleocapsid protein, NCp7, that contains a single tryptophan residue (Trp37) within a retroviral zinc finger sequence. Binding of poly(A) or poly(U) to the tetrapeptides induces larger delta E(0,0) and delta D than when bound to NCp7, indicating stronger stacking interactions. Poly(I), on the other hand, produces larger shifts in Trp37 of NCp7. Binding of rG(8) produces sequence-dependent effects in the peptides. When bound to NCp7, but in contrast with tetrapeptide binding, nucleic acids produce large changes in triplet state kinetics consistent with enhanced spin-orbit coupling. These results are discussed in terms of three limiting types of tryptophan-base interaction: intercalation, aromatic stacking, and edge-on interaction. These should have differing effects on the properties of the triplet state.

Ligand-induced conformational changes in lactose repressor: a phosphorescence and ODMR study of single-tryptophan mutants.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations.

Phosphorescence and optically detected magnetic resonance characterization of the environments of tryptophan analogues in staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on the triplet states of the tryptophan analogues, 7-azatryptophan (7AW), 5-hydroxytryptophan (5HW), and 4-, 5-, and 6-fluorotryptophan (4FW, 5FW, 6FW), when incorporated at position 140 of wild-type Staphylococcal nuclease (7AW-nuclease, etc. ), positions 66 and 140 of its V66W mutant (7AW-V66W, etc.), and the deletion fragment of the latter, Delta 137-149 (7AW-V66W', etc.). These measurements point to the retention of protein structure at position 140 in each of the wild-type nuclease analogues. Substitution of the analogue at both tryptophan sites of V66W leads to structured sites with differentiated triplet-state properties for all analogues except 7AW-V66W, whose structure is destabilized. 5HW-V66W' is the only fragment that apparently lacks structure at position 66. All other V66W' analogues exhibit a structured environment at position 66 (4FW-V66W' was not studied), but in each case this site can be differentiated readily from the corresponding site in intact V66W. 7AW-V66W' is resolved by ODMR into two discrete structures with slightly differing zero field splittings (ZFS). Interaction of the protein with 5HW at position 66 of 5HW-V66W induces a 2-fold increase in the ZFS E parameter, which is reduced to its normal value upon formation of the fragment, 5HW-V66W'. Analogous effects occur for 5FW, but on a smaller scale.

Study of complexes of a tryptophan-free mutant of E. coli trp aporepressor with tryptophan analogues using optically detected magnetic resonance (ODMR).

Phosphorescence and optically detected magnetic resonance (ODMR) spectra of tryptophan (W) and several of its analogues (4-, 5-, 6-methyltryptophan (MeW); 4-, 5-, 6-fluorotryptophan (FW); 5-bromotryptophan) are compared with those of complexes formed with the W-free trp aporepressor from Escherichia coli (W19,99F). W19,99F binds W and each analogue except 4-FW with an estimated KD < or = 30 microM; triplet state spectroscopic and kinetic effects that accompany binding at the corepressor site are reported. ODMR data for the MeW isomers are presented for the first time. No binding of 7-azaW is observed, in agreement with the low affinity found by previous workers.

Binding of the nucleocapsid protein of type 1 human immunodeficiency virus to nucleic acids studied using phosphorescence and optically detected magnetic resonance.

The binding of p7 nucleocapsid protein of type 1 human immunodeficiency virus (HIV-1) to various oligonucleotides and polynucleotides has been investigated by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The intrinsic spectroscopic probe used in these studies is the photoexcited triplet state of Trp37, which is associated with the C-terminal zinc finger of p7 and is its only tryptophan residue. Complex formation produces a red-shift of the phosphorescence 0, 0-band (DeltaE0,0) of Trp37 as well as a reduction of the zero field splitting (zfs) D parameter. Increases of -DeltaE0,0 (A < C < U < G

Analysis of DNA-protamine interactions by optical detection of magnetic resonance.

Optically detected magnetic resonance (ODMR) has been used to identify the binding site of a synthetic protamine subdomain to the major groove of DNA. A 14 amino acid peptide (R6WGR6) analog of the central DNA binding domain of bull protamine was synthesized with phenylalanine replaced by tryptophan (Trp). The peptide was bound to double-stranded poly(dABrdU) and to calf thymus DNA (CT DNA) and the complexes characterized as "wet" solids using ODMR techniques. The appearance of the D + E transition in the slow passage ODMR and of short-lived components in the phosphorescence decay of the complex of R6WGR6 with poly(dABrdU) is diagnostic of a heavy atom effect. This can only occur if the peptide binds in the major groove of poly(dABrdU). The microenvironment of Trp in the nucleoprotein complex was characterized by phosphorescence, radiative decay lifetimes, and low-temperature ODMR measurements before and after binding to DNA. Bathochromic shifts in the phosphorescence emission upon exciting to the red in CT DNA-peptide suggest that the Trp is in a polar environment, while the red-shifted position of the 0, 0-band emission points to a more polarizable environment. The heavy atom effect strongly suggests a Trp location within the major groove of DNA. A partial stacking of Trp with the polarizable nucleobases and simultaneous interactions with the phosphate-guanidinium ion pairs and/or water molecules in the major groove of DNA which might not be totally displaced upon binding of the peptide could explain this conflicting evidence. Extrapolation of results from the system studied to protamine binding in sperm chromatin strongly suggests that the predominant binding site of protamine is the major groove of DNA.

Fluorescence, phosphorescence, and optically detected magnetic resonance studies of the nucleic acid association of the nucleocapsid protein of the murine leukemia virus.

Fluorescence, phosphorescence, and optical detection of triplet state magnetic resonance (ODMR) are employed to investigate the interaction of p10, the nucleocapsid protein of the Moloney murine leukemia virus, with nucleic acids. p10 is a 55-amino acid protein containing a single zinc finger motif, C26C29H34C39, that includes Y at position 28 and W at position 35. In addition, the interactions of a zinc finger peptide, p10-ZF, comprising residues 24-41 of p10, and a doubly mutated 24-41 peptide, p10-ZF' in which the positions of Y and W are interchanged, also are reported. The measurements focus on the direct involvement of the sole W residue in the nucleic acid interaction. Fluorescence quenching and salt-back titrations indicate complex formation of p10 with several octanucleotides--(dT)8, (dI)8, (dU)7dT, and (5-BrdU)7dT--and with the polynucleotides poly(dT) and poly(dI). Poly(dI) binds with the highest affinity. Apparent binding constants and salt-back midpoints are reported. Neither p10-ZF nor p10-ZF' exhibits significant fluorescence quenching by these DNA substrates. Binding of p10-ZF to fluorescent poly(ethenoadenylic acid) was detected with greatly reduced affinity relative to p10, but binding of p10-ZF' was undetectable. These results are in general agreement with phosphorescence and ODMR measurements monitoring W. Addition of poly(I) to p10 leads to a phosphorescence red shift, reduction in the zero-field splitting (ZFS) parameters D and E, and a significantly reduced phosphorescence lifetime, each consistent with aromatic stacking interactions between W and the nucleobases. These effects are smaller with p10-ZF and undetectable with p10-ZF'. Poly(U) produces no significant changes in the triplet state parameters of W; no stacking interactions are observed even for p10. (5-BrdU)7dT yields large phosphorescence red shifts in p10 and p10-ZF, and reductions of D, but no significant heavy atom effects. These effects probably are due to enhanced local polarizability caused by Br, but any stacking interactions in these complexes would exclude van der Waals contacts between W and the Br atoms.

Thermodynamics of the unfolding and spectroscopic properties of the V66W mutant of Staphylococcal nuclease and its 1-136 fragment.

Spectroscopic studies have been performed to characterize the solution structure of the V66W mutant of Staphylococcal nuclease and the corresponding 1-136 fragment, referred to as V66W'. Whereas wild-type nuclease has a single tryptophan residue at position 140, the V66W mutant has a second tryptophan residue at position 66, which is the only such residue in V66W'. Steady-state and time-resolved fluorescence studies show Trp-66 in V66W' to have a blue emission, a relatively large fluorescence quantum yield, a long lifetime, a significant degree of protection from solute quenchers, and to depolarize with a relatively long rotational correlation time. These results characterize Trp-66 in V66W' as being a buried residue, which indicates that this fragment retains some global structure. Circular dichroism (CD) data are consistent with the fragment having lost most of the alpha-helical content of the wild type, while retaining beta-sheet structure. The CD spectrum in the aromatic region also suggests that Trp-66 in the fragment experiences an asymmetric environment, which is not identical to that in the full length mutant, V66W. In addition, optical detection of triplet state magnetic resonance (ODMR) spectroscopy can clearly resolve the tryptophan residues and demonstrates differences between the local environment of Trp-66 in V66W and in V66W', as well as small differences in the Trp-140 environment in wild type and in V66W. Guanidine-HCl induced and thermally induced unfolding studies were performed by simultaneously acquiring CD and fluorescence data as a function of the perturbation and then performing a global analysis of such multiple data sets in terms of two-state and three-state unfolding models. Whereas data for wild-type nuclease and the V66W' fragment are well characterized by a two-state unfolding model, data for the V66W mutant are better characterized by a three-state process. That is, both the denaturant- and temperature-induced unfolding of V66W involves the significant population of an equilibrium unfolding intermediate. Our global analyses yield thermodynamic parameters for the unfolding transitions, and we show that the data for V66W can be described by a constrained three-state model in which the transition of the intermediate to the fully unfolded state is fixed to have the same thermodynamic parameters that describe the unfolding of the V66W' fragment.

Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant.

The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L.E., & Maki, A.H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor.

Phosphorescence and optically detected magnetic resonance investigation of the binding of the nucleocapsid protein of the human immunodeficiency virus type 1 and related peptides to RNA.

The RNA and DNA complexes of nucleocapsid protein p7.Zn (NCp7.Zn) of the human immunodeficiency virus type 1 (HIV-1) are studied by phosphorescence and optically detected magnetic resonance (ODMR). The single tryptophan, Trp37, which is located on the C-terminal zinc finger domain is used as an intrinsic probe. Reductions in the triplet state zero-field splitting (zfs) D parameter of Trp37 upon complex formation with poly(I) and poly(U) are observed. These results, in conjunction with the phosphorescence red-shifts and triplet state lifetime reductions that are observed, suggest the presence of aromatic stacking interactions between NCp7.Zn and the bases of the RNA polymers. An alteration of the intersystem crossing pattern upon complex formation, in addition to the above mentioned spectroscopic shifts, also is consistent with previously observed tryptophans that undergo stacking interactions with DNA bases [Zang, L.-H., Maki, A.H., Murphy, J.B., & Chase, J.W. (1987) Biophys. J. 52, 867-872. Tsao, D.H.H., Casas-Finet, J.R., Maki, A.H., & Chase, J.W. (1989) Biophys. J. 55, 927-936]. These conclusions support those from a recent ODMR study [Lam, W.-C., Maki, A.H., Casas-Finet, J.R., Erickson, J.W., Sowder, R.C., II, & Henderson, L.E. (1993) FEBS Lett. 328, 45-48] of NCp7.Zn binding to 5-mercurated polyuridylic acid [poly(5-HgU)] in which stacking interactions between the RNA and NCp7.Zn are inferred from the observation of an external heavy atom effect induced on Trp37.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformer interconversion in the LC analysis of triostin A and its under-N-methylated synthetic analogue.

Multiple peak formation and interconversion in the liquid chromatographic (LC) analysis of triostin A and its under-N-methylated synthetic analogue, [N-MeCys3,N-MeCys7]-TANDEM (MCTANDEM), are investigated as a function of column temperature. Slow interconversion between chromatographic peaks, ascribed to the presence of the n- and p-solution conformers of the peptides, is exhibited in the normal-phase elution profiles of triostin A and in the reversed-phase elution profiles of MCTANDEM. A chromatographic model is developed to estimate the kinetics of conformer interconversion. Reversed-phase LC analysis of the n- and p-conformers of MCTANDEM yields a value of 0.01/s for the apparent interconversion rate constant (kn-p) at 25 degrees C, with a corresponding activation energy of 16 kcal/mol. Normal-phase LC analysis of the n- and p-conformer interconversion of triostin A dissolved in chloroform results in a value of 0.04/s for kn-p at 25 degrees C, with a corresponding activation energy of 18 kcal/mol. For triostin A, normal-phase LC findings as a function of column temperature are compared with 1H nuclear magnetic resonance (NMR) line-width measurements between 80 degrees C and 140 degrees C for the n- and p-conformers, which yield an activation energy of 19 kcal/mol and an extrapolated value of 0.02/s at 25 degrees C for kn-p in deuteriochloroform.

Study ofL-tryptophan corepressor binding to mutatedE. coli tryptophan repressor proteins by optically detected triplet-state magnetic resonance.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements have been carried out on the tryptophan (Trp) residues ofEscherichia coli Trp repressor protein (W Rep) and its two single Trp-containing mutants, W19F and W99F. The enhanced resolution afforded by the W19F and W99F mutants allowed us to characterize the triplet state of boundL-Trp corepressor using phosphorescence wavelengt-selected ORMR spectroscopy. We find that at 77 K the 0,0 band peak wavelength ofL-Trp is shifted from 405.5 nm in the aqueous solvent to ca. 410 nm when bound to the corepressor binding site. This red shift of the phosphorescence along with a corresponding increase in the zero-field splittingE value and narrowing of the ODMR linewidth characterize a binding site that is less polar, as well as more polarizable and homogeneous, than the aqueous solvent. This conclusion is in agreement with the X-ray crystallographic structure of the holorepressor protein that places the indole chromophore of the bound corepressor in a cleft in which it is sandwiched by the side chains of arginines 54 and 84.

Characterization of the two tryptophan residues of the lactose repressor from Escherichia coli by phosphorescence and optical detection of magnetic resonance.

The native lactose repressor from Escherichia coli (Lac Rep) and two single-point mutants, W220Y and W201Y, were investigated using low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Emission from two tryptophan residues was evident in the phosphorescence spectrum of native Lac Rep at 77 K. Using the single-point mutants, the triplet-state properties of tryptophans 201 and 220 were obtained independently. Trp 220 was characterized as a partially solvent-exposed residue (0,0 band centered at 409.5 nm), while tryptophan 201 exhibited the properties of a buried residue (0,0 band centered at 413.5 nm). Both single-point mutant proteins experienced changes in tryptophan triplet-state properties as a result of binding either of two inducer sugars: isopropyl beta-D-thiogalactoside, a monosaccharide, or melibiose, a disaccharide. Putative singlet-singlet energy transfer from tryptophan 220 to tryptophan 201 was also investigated, but the quantitative results must be viewed with some caution.

Luminescence studies with trp repressor and its single-tryptophan mutants.

Time-resolved and steady-state fluorescence, low-temperature phosphorescence, and optically detected magnetic resonance (ODMR) measurements have been made to resolve the luminescence contributions of the two intrinsic tryptophan residues in the subunits of trp aporepressor from Escherichia coli. Assignments of spectral information have been confirmed by use of the single-tryptophan mutants W19F and W99F. Solute fluorescence quenching studies show that both Trp19 and Trp99 are exposed to acrylamide and iodide, with Trp99 being the more exposed. Time-resolved and steady-state fluorescence measurements show Trp19 to have a bluer emission, a longer mean fluorescence decay time, a higher quantum yield, and essentially no independent rotational motion with respect to the protein. Trp99 is found to have a redder emission, a shorter mean fluorescence decay time, a lower quantum yield, and a significant degree of rotational freedom. Phosphorescence studies show a clear resolution of 0-0 vibronic transitions for each type of residue, with maxima at 407 and 415 nm that are assigned to Trp19 and Trp99, respectively. ODMR measurements show the zero-field splitting parameters to be quite characteristically different for each tryptophan residue. The existence of resonance energy transfer from Trp19 to Trp99, in the wild-type protein, is indicated by three types of data: comparison of the long-lived decay time (attributed to Trp19) in the absence (W99F) and presence (wild type) of the acceptor Trp99, comparison of the fluorescence quantum yield of the wild-type and mutant proteins, and deviations from the expected phosphorescence intensities for Trp19 and Trp99 in the absence of energy transfer.

Evidence for stacking interactions between 5-mercurated polyuridylic acid and HIV-1 p7 nucleocapsid protein obtained by phosphorescence and optically detected magnetic resonance (ODMR).

The photoexcited triplet state of Trp-37 in the C-terminal zinc finger of the HIV-1 p7 nucleocapsid protein was used as a probe of p7 interactions with the heavy atom-derivatized RNA homopolymer, poly-5-mercuriuridylic acid (5-HgU). Binding of p7 to 5-HgU (Hg blocked with 2-mercaptoethanol) produces an external heavy atom effect (HAE) on Trp-37 characterized by fluorescence quenching, reduction of the phosphorescence lifetime by three orders of magnitude, and the appearance of the D+E phosphorescence-detected ODMR signal, absent in unperturbed Trp, but induced by a HAE. The details of the HAE are consistent with out-of-plane van der Waals contact of Hg with the indole chromophore of Trp-37. Steric requirements suggest further that the Trp-RNA contact occurs via an aromatic stacking interaction.

Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme.

The radiative quantum yield, phi op, of the triplet state of human alpha-lactalbumin (HLA) has been measured in the temperature range between 6 K and the softening point of the aqueous glass (approximately 150 K). phi op has little temperature dependence below approximately 30 K, but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-state model in which the phosphorescence originates primarily from a donor, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy transfer process. The model assumes that the acceptor (Trp 60) triplet state undergoes radiationless deactivation by a proximal disulfide residue, while the donor (Trp 104) has no such extrinsic quencher. The decrease of phi op with increasing temperature is accounted for by the thermally activated triplet-triplet energy transfer process. The disulfide quenching rate constant itself is assumed to be temperature independent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphorescence quenching in HLA occurs with an activation energy of 97 cm-1, which we associate with a barrier to the energy transfer process. The data are fit well by the model if we assume a value for the temperature-independent disulfide quenching constant of kQ > 3 s-1 that is consistent with recent measurements on indole-disulfide model systems (Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). Similar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calculations since it is the simplest, lacking Trp 26, a residue not considered in the model, that probably contributes significantly to the phosphorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPLA, which contains Trp 104 but lacks Trp 60, shows qualitatively less thermally induced phosphorescence quenching than HLA, BLA, and HEWL, thus supporting the postulated quenching model.