Jeanne R. Small

Analysis of photoacoustic waveforms using the nonlinear least squares method

Pulsed-laser photoacoustics is a technique which measures photoinduced enthalpic and volumetric changes on the nano- and microsecond timescales. Analysis of photoacoustic data generally requires deconvolution for a sum of exponentials, a procedure which has been developed extensively in the field of time-resolved fluorescence decay. Initial efforts to adapt an iterative nonlinear least squares computer program, utilizing the Marquardt algorithm, from the fluorescence field to photoacoustics indicated that significant modifications were needed. The major problem arises from the wide range of transient decay times which must be addressed by the photoacoustic technique. We describe an alternative approach to numerical convolution with exponential decays, developed to overcome the problems. Instead of using an approximation method (Simpsons rule) for evaluating the convolution integral, we construct a continuous instrumental response function by quadratic fitting of the discrete data and evaluate the convolution integral directly, without approximations. The success and limitations of this quadratic-fit convolution program are then demonstrated using simulated data. Finally, the program is applied to the analysis of experimental data to compare the resolution capabilities of two commercially available transducers. The advantages of a broadband, heavily damped transducer are shown for a standard organic photochemical system, the quenching of the triplet state of benzophenone by 2,5-dimethyl-2,4-hexadiene.

Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump.

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.

Kinetics of Local Helix Formation in Poly-L-Glutamic Acid Studied by Time-Resolved Photoacoustics: Neutralization Reactions of Carboxylates in Aqueous Solutions and Their Relevance to the Problem of Protein Folding

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.

An experimental methodology for measuring volume changes in proton transfer reactions in aqueous solutions

A fast perturbation in proton concentration can be induced in aqueous solution using a pulsed ultraviolet laser and suitable photolabile compounds which, upon photoexcitation, irreversibly release protons. The volume change and the rate constant for the reaction of the photodetached protons with proton-accepting groups in solution can be monitored using time resolved photoacoustics. A typical proton concentration jump of 1 microM can be obtained with a 200-microJ laser pulse at 308 nm. Reaction dynamics from 20 ns to 5 micros can be easily followed. The methodology we establish represents a direct, time-resolved measurement of the reaction volume in proton transfer processes and an extension to the nanosecond-microsecond range of traditional relaxation techniques, such as stopped-flow. We report example applications to reactions involving simple molecules and polypeptides.

Combined photoacoustic and fluorescent quenching studies on organic dyes

The development of deconvolution techniques in pulsed-laser, time-resolved photoacoustics has opened the possibility of accurately distinguishing between processes occurring on different time scales, and has given photoacoustics better resolution in determining reaction enthalpies and quantum yields. While fluorescent signals are usually generated by a single de- excitation pathway in the fluorophore, photoacoustic signals usually arise from different sources, such as excited singlet and triplet deactivation, occurring on well-distinguished time scales. The understanding of the effect of quenching on photoacoustic signals therefore requires careful analysis of the data. In this work, a model is developed to describe the effect of fluorescence quenching on photoacoustic signals. The model takes advantage of the time resolution in pulsed-laser photoacoustics. Both static and dynamic quenching are taken into account. Important photophysical parameters (fluorescence and intersystem crossing quantum yields, the bimolecular quenching rate constant, and the volume of the sphere of action) appear in the expressions describing the dependence of photoacoustic signal on quencher concentration. Data from both steady-state fluorescence and time-resolved photoacoustic quenching measurements are analyzed simultaneously using a set of equations containing common parameters. Experimental data on the quenching of organic dyes are presented which support the validity of the model.

Fluorescence anisotropy decays with minimal instrumental artifacts

We have constructed a fluorescence decay instrument with computer-automated optical components and data acquisition. Stepping motors control polarizer orientations, filter holders, retarder for incident intensity control, and four position sample changer. The changer uses a mechanical indexer for rapid, precise positioning, and has thermoelectric temperature control. Nitrogen flush and magnetic stirring are provided for all four cuvette positions. An automatic shutter protects the photomultiplier tube, closing automatically when the instrument is opened or the measured photon flux exceeds a predetermined limit. Optical sensors relay position information to the computer for all moving components. The instrument is controlled by a Windows-based program designed to accommodate users of widely varying ability. An inexperienced student can automatically run a complex anisotropy decay experiment with careful sensitivity corrections. Using simple editing functions, a more experienced user, on the other hand, can vary an experiment in minute detail. Automatic algorithms are used to home the instrument at the beginning of an experiment, to increase incident laser intensity until a specified count rate is achieved, and to maintain the count rate during a measurement. We also summarize here some instrumental artifacts common to time-resolved fluorescence data as well as approaches we have used to minimize their effects on recovered decay parameters.