DETECTION OF WATER PROXIMITY TO TRYPTOPHAN RESIDUES IN PROTEINS BY SINGLE-PHOTON RADIOLUMINESCENCE

We recently developed a single photon radioluminescence (SPR) techniqu e to measure submicroscopic distances in biological samples [Bicknese et al., and Shahrokh et al., Biophys. J., 63 (1992) 1256-1279]. SPR ar ises from the excitation of a fluorophore by the energy deposited from a slowing beta decay electron. The purpose of this study was to detec t (H2O)-H-3 molecules near tryptophan residues in proteins by tryptoph an SPR. To detect small SPR signals, a sample compartment with reflect ive ellipsoidal optics was constructed, and amplified signals from a c ooled photomultiplier were resolved by pulse-height analysis. A Monte Carlo calculation was carried out to quantify the relationship between SPR signal and (H2O)-H-3-tryptophan proximity. Measurements of trypto phan SPR were made on aqueous tryptophan; dissolved melittin (containi ng a single tryptophan); native and denatured aldolase; dissolved aldo lase, monellin, and human serum albumin; and the integral membrane pro teins CHIP28 (containing a putative aqueous pore) and MIP26 using (H2O )-H-3 or the aqueous-phase probe H-3-3-O-methylglucose (OMG). After su btraction of a Bremsstrahlung background signal, the SPR signal from a queous tryptophan (cps .mu Ci(-1) mu mol(-1) +/- SE) was 8.6 +/- 0.2 w ith (H2O)-H-3 and 7.8 +/- 0.3 with (3)HOMG (n = 8). With (H2O)-H-3 as donor, the SPR signal (cps .mu Ci(-1) mu mol(-1)) was 9.0 +/- 0.3 for monomeric melittin in low salt (tryptophan exposed) and 4.6 +/- 0.8 (n = 9) for tetrameric melittin in high salt (tryptophans buried away fr om aqueous solution). The ratio of SPR signal obtained for aldolase un der denaturing conditions of 8 M urea (fluorophores exposed) versus no n-denaturing buffer (fluorophores buried) was 1.53 +/- 0.07 (n = 6). R atios of SPR signals normalized to fluorescence intensities for monell in, aldolase, and human serum albumin, relative to that for d-tryptoph an, were 1.42, 1.09, and 1.04, indicating that the cross-section for e xcitation of fluorophores in proteins is greater than that for tryptop han in solution. For the CHIP28 and MIP26 proteins in membranes, the r atio of SPR signal obtained with (H2O)-H-3 versus (3)HOMG was 1.35 +/- 0.13 (CHIP28, n = 5) and 0.99 +/- 0.02 (MIP26). These data are consis tent with the existence of an aqueous channel through CHIP28 that excl udes small solutes. We conclude that tryptophan radioluminescence in p roteins is measurable and provides unique information about the presen ce of local aqueous compartments.