Inhibition of luciferase activity by 3-azioctanol
In order to assess whether 3-azioctanol, like octanol and other alkanols, inhibits luciferase, American firefly luciferase (20 nM) was pre-incubated for 15 minutes with 0 to 6000 µM 3-azioctanol in the presence of 28 µM luciferin at 22±1°C. These samples were then rapidly mixed with 4 mM ATP in the stopped flow spectrofluorimeter. Final concentrations were half those given above. The light emitted was monitored for 5 s. It showed an initial lag of ~50 ms, rose to a peak at ~300 ms and fell slowly for 5 s (). Increasing concentrations of 3-azioctanol reduced the maximum light output without altering the overall pattern of time–dependence. We used the slope of the linear region of the kinetic curve (~65 to ~200 ms) as a measure of inhibition. The data were normalized between the control and 3000 µM slopes and fitted to a logarithmic equation by nonlinear least squares () to yield an IC50
of 200±46 µM and a slope of 1.0±0.2. For comparison, the general anesthetic EC50 for 3-azioctanol is 160 µM 
. Under similar conditions, 225 µM 1-octanol inhibited luciferase by ~50%. This compares to a reported IC50
of 280 µM for 1-octanol 
. 3-Azibutanol (1 mM) also inhibited luciferase. Thus, azialkanols behave like other 1-alkanols with respect to inhibition of luciferase.
The 3-Azioctanol inhibits ATP-induced luciferase activity.
Equilibrium photolabeling of luciferase by [3H]3-azioctanol
Because the inhibition of luciferase activity by alcohol is ATP-dependent 
, we determined the effect of ATP on the photoincorporation of [3
H]3-azioctanol. Increasing concentrations of [3
H]3-azioctanol were preincubated for 30 min with 4.3 µM Japanese firefly luciferase in the absence or presence of 2 mM ATP, and photolabeled as described above. In the presence of ATP, the level of photoincorporation increased linearly (), consistent with either a nonspecific action or binding to sites with very low affinity. In the absence of ATP, photoincorporation was higher at all alcohol concentrations and the shape of the curve suggested saturable binding with a dissociation constant above 1 mM. Similar data were obtained with American firefly luciferase. Thus, at equilibrium, ATP inhibits photoincorporation of [3
H]3-azioctanol into firefly luciferase.
ATP modulates the photoincorporation of [3H]-3-Azioctanol into luciferase at equilibrium.
Time resolved photolabeling with [3H]3-Azioctanol
At first sight the ATP-induced decrease in photoincorporation of [3
H]3-azioctanol observed above contradicts the allosteric hypothesis of Moss et al 
, which predicts that ATP induces a higher affinity binding of alcohols. However, their hypothesis refers to the initial phase of the reaction (≤300 ms), not at equilibrium. Accordingly, we determined photoincorporation into luciferase 200 ms after the addition of 2 mM ATP using time resolved photolabeling. Because of the large sample size required by the apparatus and the cost of tritiated ligands, only a single concentration of [3
H]3-azioctanol (1 µM) was examined. The data presented in (inset) show that, unlike at equilibrium, ATP doubles the photoincorporation of [3
H]3-azioctanol into luciferase at 200 ms. Analysis of the data by the Student's t-test yielded a p-value of 0.002 indicating that the difference in photoincorporation observed without ATP (white bar) and with ATP (black bar) is statistically significant.
Identification of residues photolabeled in luciferase
To identify residues in the anesthetic binding site(s), we photolabeled Japanese luciferase in both the presence and absence of ATP (at 200 ms and at equilibrium) with the diazirinyl n-alkanols (3-azibutanol, 3- and 7-azioctanol) and with TFD-benzyl alcohol. All samples were proteolytically digested and subjected to HPLC-MSMS. We were able to sequence 94% of the Japanese luciferase, but our data (see below and & ) show that photolabeling is confined to a few major peptides together with a number of nonspecific sites. Furthermore, to enhance our ability to map the binding sites we used both aromatic and aliphatic alcohols containing diazirine groups because these two classes of agent have different photoselectivities. Although no formal survey has been published, aromatic and aliphatic diazirines react selectively with nearly complementary sets of residues (roughly Leu, Ile, Ser, Met Val, Phe vs. Tyr, Glu, Asp, His, Met, respectively) 
Residues photolabeled in a tryptic fragment of Japanese Firefly Luciferase (306-YDLSNLVEIASGGAPLSK-323)a identified by LTQ–FT mass spectrometry and MSMS.
Residues photolabeled in a tryptic fragment of Japanese Firefly Luciferase (340-QGYGLTETTSAIIITPEGDDKPGASGK-366)an identified by LTQ–FT mass spectrometry and MSMS.
We also photolabeled the American firefly luciferase at equilibrium with the azialkanols, and found the labeling pattern to be identical. The American and Japanese firefly luciferases have both been used in anesthetic studies; the American has been studied extensively, but the highest resolution structure is for the Japanese. Overall, these two proteins are highly homologous (67%), and we have used the Japanese numbering system in this discussion (for the residues of interest, subtracting 2 from the Japanese residue number usually gives that of the American). The two luciferases sometimes differ in their proteolytic cleavage patterns, and we have found this useful (see below).
Residues photolabeled on peptide Tyr-306– Lys-324
The Japanese luciferase was exposed to ATP and 100 µM 3-azioctanol for 200 ms, rapidly frozen and photolabeled. The samples were treated as described in Methods
, and the tryptic fragments analyzed by HPLC-MSMS. A doubly charged peptide of mass 1963.1679 Da with a retention time of 29.1 min was detected by FTMS. This peptide was identified as the 18–residue peptide YDLSNLVEIASGGAPLSK beginning at Y306 and incorporating a single molecule of azioctanol. It was fragmented by collision with an inert gas to yield peptide fragments with an intact N-terminus, called b-ions, or an intact C-terminus, called y-ions. shows the MSMS spectrum that was used to identify the point of photoincorporation as Glu-313. A sequence of mostly strong singly charged y-ions from y15 to y4 was observed with a loss of 3-azioctanol between y11 and y10. This conclusion was consistent with the b-ion pattern where a run of peaks was observed from b14 to b6 (except b12), with loss of 3-azioctanol between b8 and b7. This assignment had an Xcorr
value of 5.9 (). Similar results were obtained for Japanese luciferase labeled with 100 µM 7-azioctanol and 1 mM 3-azibutanol at equilibrium in the absence of ATP (; Figs. S1
). The percent of observed peptides that were labeled was 25–50% at 200 ms after adding ATP, and simultaneous addition of luciferin reduced this to 0–10%. At equilibrium, photolabeling of Glu-313 could be resolved at concentrations as low as 10 µM 3-azioctanol (Fig. S3
) and 100 µM 3-azibutanol.
Identification of photolabeled residues in the peptide Tyr-306– Lys-324.
When Japanese luciferase was photolabeled with 100 µM TFD-benzyl alcohol after incubation for 200 ms with ATP, a major photolabeled trypsin product that eluted from the HPLC at 29.3 min was a doubly charged peptide of mass 2021.9993 Da. It was identified as an 18–residue peptide YDLSNLVEIASGGAPLSK, starting at Y306, photolabeled by a single TFD-benzyl alcohol. shows the MSMS spectrum that was used to identify the point of photoincorporation as Ser-316. A sequence of mostly strong singly charged y-ions from y15 to y4 with the exception of y7 was observed with a loss of TFD-benzyl alcohol between y8 and y6. This result is consistent with the b-ion pattern where a run of peaks was observed from b17 to b5 with the exception of b11, with loss of TFD-benzyl alcohol between b12 and b10. Thus, the photolabeled residue is Ser-316. This assignment had an Xcorr value of 5.39 ().
From the same experiment, another tryptic peptide, eluting a little later (30.5 min), was doubly charged with the same mass of 2021.9993 Da, but it was not photolabeled at Ser-316. The MSMS spectrum shows a run of mostly strong singly charged y-ions from y14 to y4 with a loss of TFD-benzyl alcohol between y10 and y9, placing the site of photoincorporation at Ile-314 (). This conclusion was consistent with the b-ion pattern where a run of peaks was observed from b6 to b14, with loss of TFD-benzyl alcohol between b9 and b8. This assignment had an Xcorr value of 4.43 (). Consistent with our observation that TFD-benzyl alcohol photolabeled both Ile-314 and Ser-316, we found that double labeling predominated over single labeling by fourfold with +ATP at 200 ms, but the quality of these double labeled spectra were inadequate to resolve their sites of photoincorporation. The frequency of photoincorporation was lower than with the azialkanols, consistent with the known low photolabeling efficiency of aromatic diazirines, making it difficult to resolve small change in photoincorporation, but simultaneous addition of both ATP and luciferin completely abolished photoincorporation into both residues.
Residues photolabeled on peptide Gln-340 – Lys-366
A second peptide that was photolabeled was a 27–residue tryptic peptide starting at Gln-340. Sequencing this peptide proved to be difficult. Neither digestion by chymotrypsin nor Asp-N produced a better situation. In some cases we could not unambiguously define the point of incorporation. When Japanese firefly luciferase was photolabeled with 1 mM 3-azibutanol after incubation for 200 ms with ATP, a triply charged tryptic peptide eluted from the HPLC at 20.3 min with a mass of 2779.9283 Da by FT–MS, corresponding to a photolabeled 27–residue peptide starting at Q340—QGYGLTETTSAIIITPEGDDKPGASGK. The MSMS spectrum of this peptide is shown in . A sequence of doubly charged y-ions from y24++ to y9++, with the exception of y11++, was observed to be modified, suggesting that the fragment 358–DDKPGASGK is photolabeled. Consistent with this, a run of weaker singly charged y-ions was observed from y13 to y6, with the exception of y9. In this series all ions are modified except for y8, y7 and y6, narrowing the assignment of the photolabeled residue to Asp-358. The assignment based on the y+ ions is somewhat insecure because of their low amplitude. Thus, the evidence ruling out Glu-356 as a site of photolabeling rests on the large doubly charged y9++ and y10++ ions. The b ions had low intensity and were not used in this analysis. This assignment had an Xcorr value of 5.8. Under the same conditions when luciferin was included no photolabeled peptides were observed.
Identification of photolabeled residues in the peptide Gln-340 – Lys-366.
Tryptic digestion of Japanese luciferase photolabeled with 100 µM TFD-benzyl alcohol after incubation for 200 ms with ATP yielded a triply charged photolabeled peptide of mass 2894.3869 that eluted at 24.8 min. It was identified as the same peptide as above, starting at Gln-340, photolabeled by a single TFD-benzyl alcohol. A complete series of doubly charged y-ions from y25++ to y19++ were all modified (). Another series of y++ ions from y16++ to y12++ were unmodified. Thus, photoincorporation must be in either Ser-349 or Ala-350. This assignment is consistent with the weaker run of unmodified y+ ions from y16 to y12 and from y8 to y5. Based on a large body of photolabeling data with aromatic diazirines, in particular 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID), alanine has a very low probability of being photolabeled, so we conclude that Ser-349 is the residue photolabeled by TFD-benzyl alcohol. When luciferin and ATP were added simultaneously for 200 ms there was no photoincorporation into this peptide.
Residues photolabeled on peptide Cys-284 – Lys-299
In the time resolved experiments with 100 µM TFD–benzyl alcohol, the 16–residue peptide CTSVILVPTLFAILNK eluted at ~25 minutes as a doubly charged ion with molecular weight of 2296.1277 Da by FT–MS. Unfortunately, it proved very difficult to sequence this peptide reliably. The best sequence (Xcorr
1.75) was observed for a triply labeled peptide in a run with ATP exposure for 200 ms. In a series of doubly charged y-ions, from y15++ to y10++, with the exception of y12++, one photolabel was lost between y14++ and y13++ and a second between y13++ and y11++. The final photolabel was still attached at y6++, y5++ and y4++, but likely not at y2+ (). This data is consistent with photoincorporation at Ser-286, Ile-288 as well as at one of the C-terminal residues, 297–LNK. Of these three residues, photoselectivity favors Leu-297 and y2+ probably rules out Lys-299. This assignment of labeling was typical of many other lower quality spectra. Photoincorporation into this peptide was much more efficient (~5×) than into the peptide in the previous section. It was often observed with 2 or 3 TFD–benzyl alcohol molecules photoincorporated.
Identification of photolabeled residues in two peptides.
Residues photolabeled on peptide Asp-226 – Arg-263
This peptide was rarely seen when the Japanese luciferase was sequenced, but was observed in the American luciferase, possibly because 6 residues are not conserved. When American firefly luciferase was photolabeled with 1 mM 3-azibutanol after incubation for 200 ms with ATP, a quadruple charged tryptic peptide eluted from the HPLC at 32.5 min with a mass of 4266.1344 Da by FT–MS, corresponding to a photolabeled 38–residue peptide DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFR starting at Asp-226. This long peptide with 5 glycine and 3 proline residues was difficult to sequence entirely. A run of y-ions that all include a photolabel from y26++ to y12++ with the exception of y18++, y17++ and y14++, suggest that the photolabel is on one of the 12 residues at the N-terminal end of the peptide (). A fairly complete run of modified b-ions from b28+++ to b36+++ except b30 & 33+++ with loss of label between b32+++ and b34+++, together with unmodified b33++++ and b34++++ ions is consistent with photoincorporation on Tyr-257. Although some of these peaks are small, this conclusion is reinforced by the fact that Tyr-257 is the only residue in the N-terminal dozen residues that aliphatic diazirines have been observed to react with 
Minor labeling sites
A number of other peptides were photolabeled in an ATP–independent manner by azialkanols. These residues were invariably on the surface. For example, in a time–resolved photolabeling experiment with 1 mM 3-azibutanol in the presence of ATP and luciferin, luciferin–independent photolabeling was seen in the lid domain, primarily in Glu-499 and Tyr-304, and in a surface residue, Tyr-58. None of these residues are near the known binding sites for ATP or luciferin, and they were not photolabeled at 10 µM 3-azioctanol or 100 µM 3-azibutanol, so they represent low affinity nonspecific photoincorporation. Another surface residue, Tyr-18, was photolabeled by 1 mM 3-azibutanol in the absence but not presence of ATP. It was also observed at 10 µM 3-azioctanol and 100 µM 3-azibutanol, concentrations at which the other surface residues were not seen.
Loss of righting reflex was determined over the concentration range 5–100 µM TFD-benzyl alcohol. All animals recovered from anesthesia. The EC50 for loss of righting reflexes was 28±6.7 µM and the slope was 2.1±0.76.