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The catalytic domain of human tumor necrosis factor-α (TNF-α) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-α to generate the mature TNF-α in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-α-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 °C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 °C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

The equilibrium constants for the gas phase reactions: Cl−(HCl)n = Cl−(HCl)n−1 + HCl, (n, n−1) were measured at different temperatures with a pulsed electron beam high pressure mass spectrometer. This allowed determination of ΔGn,n−10, ΔHn,n−10, and ΔSn,n−10for reactions with n = 1 to n = 4. The enthalpy change for the reaction: (ClHCl)− = Cl− + HCl was ΔH1.00 = 23.7 kcal/mol. This value is much higher than the literature value of 14.2 kcal/mol based on Born cycles. The stabilities of the Cl−(HCl)nclusters are compared with those of OH−(H2O)nand Cl−(H2O)nmeasured earlier. It is found that the (ClHCl)−is nearly as stable as the (HOHOH)−species but that the stabilities of the higher Cl−(HCl)nclusters decreases much more rapidly than that of OH−(H2O)n. The initial strong interaction in (ClHCl) is assumed to be due to the high polarizability of Cl. For large nthis effect becomes unimportant. Cl−HOH is much more weakly bound than (ClHCl)−, however, at high nthe Cl−(H2O)ninteractions become more favorable.

By measuring the A−(H2O)n−1 + H2O = A−(H2O)nequilibria in the gas phase and their temperature dependence, the equilibrium constants and ΔHn, n–1and ΔSn, n–1for some of the hydrates of NO2−, NO3−, CN−, and OH−were determined. Available thermochemical data are used for the evaluation of the total heats of hydration of the above ions. The total heats of hydration were then compared with the ΔH1,0. Relative to the total hydration energies the ΔH1,0of the above ions were found larger than the ΔH1,0of the halide ions.An approximate linear correlation was found to exist between ΔH1,0of negative ions and the heterolytic bond dissociation energy D(A−–H+). With this relationship independent estimates for the electron affinities of NO2and NO3could be obtained.The ΔHn, n–1of OH−were found in essential agreement with earlier measurements from this laboratory and in disagreements with recent measurements (Friedman) which gave much higher values.

Determination of the temperature dependence of the equilibrium constants Kn,n−1for the reactions A −Bn = A −Bn−1 + B where A−equals Cl−and O2−and B is HOH, CH3OH, or CH3CN leads to the corresponding ΔH0n−1, ΔG0n−1,n, and ΔS0n−1,nvalues. The experimental technique is based on mass spectrometric detection of ions escaping from a high pressure ion source. At n = 1, Cl−is solvated most strongly by methanol, then CH3CN and HOH. At higher na cross over is observed with water becoming the best solvent. These results are in agreement with the positive transfer enthalpies and free energies for Cl−from the liquid solvents HOH → CH3OH and HOH → CH3CN reported in the literature.O2−is solvated more strongly than Cl−appearing thus as an ion of "size" intermediate between Cl−and F−Again CH3OH gives the highest interaction for n = 1, however for n > 1 water gives stronger interactions.

Measurements of the proton transfer equilibria: A1− + A2H = A2−with a pulsed electron beam high pressure mass spectrometer were extended to α, β, γ chlorosubstituted aliphatic acids. The equilibrium constants were used to evaluate ΔG0for the proton transfer reactions. Assuming ΔG ≈ ΔHand using standard acids AH for which the difference between the bond dissociation energy D(A—H)and the electron affinity of A, EA(A) was known one could evaluate the corresponding difference for the newly measured acids and place them on an absolute acidity scale. The gas phase acidity was observed to increase in the order: acetic, propionic, butyric, γ-Cl butyric, β-Cl butyric, β-Cl propionic, α-Cl butyric, α-Cl propionic, α-Cl acetic. The gas phase acidities are compared with those observed in aqueous solution. The effects of the Cl substituent parallel those in solution but are much larger. The attenuation occurring in solution is attributed to weaker hydrogen bonding of the chloro stabilized acid anions to water molecules.