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Expressions previously developed by Tabuchi for the velocity and absorption of sound waves in a liquid in which a reversible chemical reaction is occurring are corrected, taking into account the effect of pressure on the chemical equilibrium.

The binding studies of the interaction of ethidium bromide with DNA have been hampered by its reversibility, which prevents direct isolation and thus characterization of the complex. The recent development of photoaffinity labeling has provided a means to circumvent this problem. However, to be useful as a probe for the parent compound, a photosensitive analogue must be shown to interact in vivo and in vitro just as the parent analogue. These studies demonstrate by steady-state and nonosecond fluorescence and stopped-flow kinetics that one of the azido analogues of ethidium, 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride, binds nucleic acids quite similarly to ethidium bromide. The interaction of the other azide, 3,8-diazido-5-ethyl-6-phenyl-phenanthridinium chloride, with DNA is qualitatively different from that of the monoazide and ethidium bromide. These results suggest that the monoazide would serve as an ideal probe for determining the actual target sites of ethidium bromide in vivo and in vitro.

The separation between the two reactive thiols SH ! (Cys-704) and SH 2 (Cys-694) and that between SH t and the active site of myosin subfragment-1 were further investigated by F6rster energy transfer techniques. The SHI-SH 2 distance was determined with the probe 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1sulfonic acid (AEDANS) attached to SH 1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH z as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52 A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH I-SH 2 distance was also determined with AEDANS attached to SH ! and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH 2. The range in R for the AEDANS/DDPM pair was 12-36 ~,, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E.,Weiei, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH t and SH 2. The distance between SH 1 and the ATPase site was determined with AEDANS linked to SI-I~ 1 and the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyi)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The separation between AEDANS at SH t and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH t and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results. Abbreviations: S-1, chymotryptic subfragrnent-1 of myosin; AEDANS, 5-[[2-[(iodoacetyl)amino]ethyl]aminolnaphthalene1-sulfonic acid; IAF, 5-(iodoacetamido)fluoresi:ein; DDPM, N-(4-dimethylamino-3,5-dinitrophenyl)maleimide; TNP-ADP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); Tes, 2-[[tris(hydroxymethyl)methyllamino]ethanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; SDS, sodium dodecyl sulfate; IAA, iodoacetamide; ~-I(ADEANS, SH2), subfragment-1 modified with AEDANS at SH~ and with SH 2 unmodified; S-1(AEDANS, IAF), subfragment-1 modified with AEDANS at SH 1 and with IAF at SH2; S-I(AEDANS,DDPM)

This article deals with the use of weighted linear least squares analysis in fitting experimental results in which all of the independent and dependent variables are subject to errors of measurement.