Abstract:The stabilizing free energy of beta-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (delta delta G0 titration) was 9.51 +/- 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in bet… Show more
“…It can be seen that the value of K assoc increases with increasing concentration of ammonium acetate, reaching a maximal value at ∼10 mM. The dependence of K assoc on buffer concentration may be due to greater electrostatic shielding between trypsin, which is protonated at pH 7 (pI 10.0), and 1 + with increasing ionic strength of the solution. 3 Influence of ammonium acetate concentration on the K assoc value determined by nanoES-FT-ICR-MS for the (Tryp + 1 ) complex in an aqueous solution of trypsin (12 μM) and 1 (53 μM).…”
The interaction between the bovine pancrease trypsin (Tryp) and its competitive inhibitor benzamidine (1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study (Clark, S.M.; Konermann L. Anal. Chem. 2004, 76, 7077-7083), it was reported that the (Tryp + 1) complex could not be detected by ES-MS. Here, it is shown that, with gentle sampling conditions, it is possible to detect gaseous protonated ions of the (Tryp + 1) complex with nanoES-MS. However, the relative abundance of the detected (Tryp + 1)n+ ions is lower than expected, based on solution composition, which suggests that dissociation of (Tryp + 1)n+ ions occurs during MS sampling. The dissociation pathways and corresponding Arrhenius parameters for the protonated (Tryp + 1)n+ ions, at n = 7-9, are determined from time-resolved thermal dissociation experiments, implemented with the blackbody infrared radiative dissociation technique. The gaseous (Tryp + 1)n+ ions are found to have short lifetimes, e.g., <0.6 s, at temperatures of >100 degrees C. The use of solution additives, including polyols, carbohydrates, amino acids, and small organic molecules, to stabilize the (Tryp + 1)n+ ions during nanoES-MS analysis is investigated. Notably, the addition of imidazole to the nanoES solution is shown to preserve the (Tryp + 1)n+ ions. The Kassoc value, (1.9 +/- 0.2) x 104 M-1, determined for the (Tryp + 1) complex by the direct ES-MS method is in agreement with values determined by other analytical methods. The stabilizing effect of imidazole in nanoES-MS is further demonstrated for the interaction between carbonic anhydrase II and 5-(dimethylamino)naphthalene-1-sulfonamide. The stabilizing effect of imidazole may be due to enhanced evaporative cooling achieved by the dissociation of molecules of imidazole, bound nonspecifically, from the protein-ligand complex in the ion source.
“…It can be seen that the value of K assoc increases with increasing concentration of ammonium acetate, reaching a maximal value at ∼10 mM. The dependence of K assoc on buffer concentration may be due to greater electrostatic shielding between trypsin, which is protonated at pH 7 (pI 10.0), and 1 + with increasing ionic strength of the solution. 3 Influence of ammonium acetate concentration on the K assoc value determined by nanoES-FT-ICR-MS for the (Tryp + 1 ) complex in an aqueous solution of trypsin (12 μM) and 1 (53 μM).…”
The interaction between the bovine pancrease trypsin (Tryp) and its competitive inhibitor benzamidine (1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study (Clark, S.M.; Konermann L. Anal. Chem. 2004, 76, 7077-7083), it was reported that the (Tryp + 1) complex could not be detected by ES-MS. Here, it is shown that, with gentle sampling conditions, it is possible to detect gaseous protonated ions of the (Tryp + 1) complex with nanoES-MS. However, the relative abundance of the detected (Tryp + 1)n+ ions is lower than expected, based on solution composition, which suggests that dissociation of (Tryp + 1)n+ ions occurs during MS sampling. The dissociation pathways and corresponding Arrhenius parameters for the protonated (Tryp + 1)n+ ions, at n = 7-9, are determined from time-resolved thermal dissociation experiments, implemented with the blackbody infrared radiative dissociation technique. The gaseous (Tryp + 1)n+ ions are found to have short lifetimes, e.g., <0.6 s, at temperatures of >100 degrees C. The use of solution additives, including polyols, carbohydrates, amino acids, and small organic molecules, to stabilize the (Tryp + 1)n+ ions during nanoES-MS analysis is investigated. Notably, the addition of imidazole to the nanoES solution is shown to preserve the (Tryp + 1)n+ ions. The Kassoc value, (1.9 +/- 0.2) x 104 M-1, determined for the (Tryp + 1) complex by the direct ES-MS method is in agreement with values determined by other analytical methods. The stabilizing effect of imidazole in nanoES-MS is further demonstrated for the interaction between carbonic anhydrase II and 5-(dimethylamino)naphthalene-1-sulfonamide. The stabilizing effect of imidazole may be due to enhanced evaporative cooling achieved by the dissociation of molecules of imidazole, bound nonspecifically, from the protein-ligand complex in the ion source.
“…Similarly, for porcine trypsin (Uniprot accession number P00761, positions 9–231), the result is 8.26. Interestingly, higher experimental values of 10.0/10.5 [ 31 , 32 ] were published for the bovine enzyme and 10.2/10.8 for the porcine enzyme [ 33 , 34 ]. For ψ-trypsin, no experimental data on p I are available to our knowledge, but similar values could be expected.…”
Section: Molecular Properties and Structure Of Trypsin And Pseudotmentioning
Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.
“…The value of cH+ can be assessed from the measured pH value [20][21][22][23], from the volume of titrant of known concentration at the given pH [24][25][26][27][28][29], using both measured pH and titrant volume [10], or cell potential and titrant volume [30]. The first two procedures require background electrolyte titrations, the last two can be applied in the absence of them, and the last one avoids even pH buffer calibration.…”
Graphical abstractFour-step potentiometric acid/base titration procedure of exact characterization of pHdependent surface charging of metal oxides
CalibrationsProton balance Highlights Straightforward protocol for pHdependent charge determination of oxide nanoparticles. Response of glass electrode must be tested in background electrolytes, too. Proton concentration calibration is needed to calculate surface proton balance. The primary result is merely net proton consumption, not necessarily surface excess. Surface charge density can only be obtained, if any interfering effects are excluded.
AbstractThe increased interest in architecting nanocomposites and in clarifying their impact on health and environment necessitates a standardized method of surface characterization, because the behaviour of nanoparticles is governed largely by their surface free energy and interfacial chemistry. Potentiometric acid-base titration has been used for decades to determine surface charge density of oxides, tremendously useful in understanding and modelling oxide/electrolyte interfaces. The method has solid theoretical and experimental foundation. This is a PDF file of an unedited manuscript that has been accepted for publication. Final edited form is published in Colloids and Surfaces A, 2012Surfaces A, , 414:302-313. http://dx.doi.org/10.1016Surfaces A, /j.colsurfa.2012 widely: the deviation of seven units of pH is the rule, rather than an exception. The PZC must reflect the inherent charging behaviour, linked exclusively to the protonation, and determined by the chemistry of the oxides. Assignment of PZC (just like assignment of equilibrium constant of any chemical reaction) requires strict conditions and consistent protocol. We present here such a protocol, and some examples of application. The protocol leads through the basic steps: pH buffer calibration, proton concentration calibration in the background electrolytes, and titrations of oxides. Built-in tests help to eliminate artificial side effects simultaneously from the measurement of pH and from the oxide/electrolyte interfacial equilibria. Although these may seem self-evident, literature survey proves the need to go back to basics in order to obtain precise, reliable and comparable pH-dependent charging of oxides. It is most general that the oxides contain some unrecognized surface impurities and have a strongly pH-dependent dissolution. These distort the results of titrations; their effect increases with decreasing particle size and can become dominant for nanoparticles. The protocol presented here contains the necessary feedback loops to easily recognize and exclude these side effects.
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