Raman spectra of interchanging ß-lactamase inhibitor intermediates on the millisecond time scale.

Rapid mix-rapid freeze is a powerful method to study the mechanisms of enzyme-substrate reactions in solution. Here we report a protocol that combines this method with normal (non-resonance) Raman microscopy to enable us to define molecular details of intermediates at early time points. With this combined method, SHV-1, a class A ß-lactamase, and tazobactam, a commercially available ß-lactamase inhibitor, were rapidly mixed on the millisecond time scale and then were flash-frozen by injection into an isopentane solution surrounded by liquid nitrogen. The "ice" was finally freeze-dried and characterized by Raman microscopy. We found that the reaction is almost complete in solution at 25 ms, giving rise to a major population composed of the trans-enamine intermediate. Between 25 and 500 ms, minor populations of protonated imine are detected that have previously been postulated to precede enamine intermediates. However, within 1 s, the imines are converted entirely to enamines. Interestingly, with this method, we can measure directly the turnover number of SHV-1 and tazobactam. The enzyme is completely inhibited at 1:4 ratio (enzyme:inhibitor) or greater, a number that agrees with the turnover number derived from steady-state kinetic methods. This application, employing non-intensity-enhanced Raman spectroscopy, provides a general and effective route to study the early events in enzyme-substrate reactions.

In-situ confocal Raman observation of structural changes of insulin crystals in sequential dehydration process.

In-situ confocal Raman spectroscopy combined with relative humidity (RH) control technique was used to study the sequential dehydration process of insulin crystals. By gradually decreasing the ambient RH of the insulin crystal, the content of the hydration water in the crystal was quantitatively controlled. Tyrosine (Tyr) residues were very sensitive to the micro-environmental changes, and four Raman features 828cm(-1), 852cm(-1), 1174cm(-1) and 1206cm(-1) of Tyr were employed to monitor the dehydration process. Taking advantage of the ratios I(852)/I(828) at different RH values, the mole fractions of the 'exposed' and 'buried' Tyr residues were estimated. Moreover, using the ratio I(1174)/I(1206) as an indicator of the dehydration process, three RH regions were discriminated. This is believed to imply that different types of the hydration water were lost step by step, i.e. firstly the 'second-layer' and 'first-layer' classes, then the 'contact' class, and finally, the 'inside' class. In addition, the profile of the amide I band was observed to gradually change with RH. By band fitting of the amide I region, changes in secondary structure were quantitatively determined. And the results showed that nearly 17% of α-helix converted into ß-sheet with RH decreasing from 92% to 2%.

Adsorption and desorption kinetics of water in lysozyme crystal investigated by confocal Raman spectroscopy.

Adsorption and desorption are critical to crystal engineering for the protein crystal applications. In this paper, we present a new method to study the adsorption and desorption kinetics of waters in lysozyme crystals. H(2)O and D(2)O were used as ideal indicators for the purpose. The lysozyme crystals were prepared in a complete D(2)O environment to ensure that all the water molecules in crystal were D(2)O. The H(2)O in the gas phase directly exchanges with the D(2)O in the crystal by exposing the crystal to an ambiance with saturated water vapor. Using in situ confocal Raman microscopy, H(2)O adsorption and D(2)O desorption in the lysozyme crystals were found to follow first-order kinetics. The rate constants of H(2)O adsorption and D(2)O desorption were obtained to be equal to each other. The kinetic rates were found to linearly depend on the surface area-to-volume ratio of the crystals.

Micro-Raman analysis of association equilibria in supersaturated NaClO4 droplets.

We report an in situ strategy for the quantitative analysis of association equilibria in a single NaClO(4) droplet of nanogram mass deposited on a quartz substrate. In the new approach, the single droplet was forced to enter into a supersaturated state by decreasing the relative humidity (RH) of the environment, allowing accurate control over the concentration of the solute within the droplet. An analysis of the solvated structure of the ClO(4)(-) anion with change in molar water-to-solute ratio (WSR) was performed by micro-Raman spectroscopy within the confines of a single droplet. The symmetric stretching v(1)-ClO(4)(-) band was observed to shift from a Stokes frequency of 935 to 944 cm(-1) and was accompanied by a change in the full width at half-maximum (FWHM) from 11.4 to 16.6 cm(-1) as the WSR decreased from 16.1 to 2.3. From component band analysis of the spectral range of 900-970 cm(-1), four peaks at 933.3, 938.6, 944.1 and 946.0 cm(-1) were identified and assigned to the free solvated perchlorate anion, the solvent-shared ion pair, the contact ion pair and complex ion aggregates, respectively. As expected, the signature of the free solvated ClO(4)(-) ion was observed to decrease in intensity with a decrease in RH over the full range from 94 to 27%. The intensity of the signature from solvent-shared ion pairs was observed to rise with decrease in RH from 94% to 75% before decreasing as the RH was further reduced to 27%. Signatures of the contact ion pair and of complex ion aggregates were shown to increase over the full range of RH as the RH was reduced. Based upon the Eigen mechanism, three association equilibria were used to describe the transformations between the free solvated perchlorate anion, the solvent-shared ion pair, the contact ion pair and complex ion aggregates. The overall association constant K and the stepwise association constants K(i) (i = 1 to 3) were determined separately (0.27 +/- 0.01, 0.03 +/- 0.01, 5.49 +/- 0.95, 0.76 +/- 0.06).

Micro-Raman spectroscopic observation of water expulsion induced destruction of hydrophobic clusters in crystalline lysozyme.

In this paper, three kinds of solid lysozyme samples with different water contents were investigated by confocal Raman spectroscopy. For the rod-like lysozyme crystal with highest water content, a sudden decrease of the intensity ratio of the doublet at 1338 and 1360 cm(-1) was observed when the ambient relative humidity (RH) was lower than 86%, indicating the destruction of hydrophobic clusters of lysozyme induced by the expulsion of the hydration water from the crystal. In contrast to the rod-like crystal, tetragonal crystal and floor-like precipitate with a smaller amount of water showed no change of the structures of the hydrophobic clusters when the relative humidity was decreasing. The presence of bulk water in the rod-like crystal is believed a necessary factor for the function of the hydration water which promotes the hydrophobicity of hydrophobic clusters.

Micro-Raman study on the conformation behavior of succinate in supersaturated sodium succinate aerosols.

Micro-Raman spectra of supersaturated aerosols of sodium succinate were obtained. The conformation behavior of the succinate dianion as a function of relative humidity (RH) was investigated by combining micro-Raman spectroscopy with theoretical calculations. A shoulder at 968 cm(-1) of the v(C-CO(2)(-)) band on the rise in more concentrated droplets was believed indicative of conformation transformations. The intensity ratio (I(963)/I(997)) of the v(C-CO(2)(-)) band at 963 cm(-1) to the v(C-C) band at 997 cm(-1), versus the molar water-to-solute ratio (WSR), was used to fathom the equilibrium between gauche and trans conformations. Before saturation (WSR = 25.8) for the droplets, the ratio of I(963)/I(997) retains a value of approximately 2.6 independent of WSR, indicating that the equilibrium was not disturbed in the dilute droplets. In supersaturated droplets (WSR < 25.8), however, the ratio sharply decreases from approximately 2.6 to approximately 1.1 at WSR = 9.6, which was attributed to the formation of contact ion pairs (CIPs).