Effects of pH on the kinetic reaction

Abstract

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.

From Small-Molecule Reactions to Protein Folding: Studying Biochemical Kinetics by Stopped-Flow Electrospray Mass Spectrometry

“…The data obtained are qualitatively similar to those measured in previous continuous-flow experiments (27,29). As an example, the stopped-flow ESI MS kinetics for the [hMb ϩ 14H] 14ϩ ion are depicted in Fig.…”

“…They are well described by biexponential fits with time constants of 0.33/3.1 s (optical), and 0.29/2.8 s (ESI MS). The two time constants reflect unfolding of hMb and loss of heme from the transient intermediate, respectively (27,29). The kinetics are somewhat faster than those in our previous study (27) where the reaction was studied at pH 3.2.…”

“…Unfolding disrupts the native hemeprotein interactions so that the final product of this reaction is unfolded apo-myoglobin (aMb). Using ESI MS combined with continuous-flow mixing we have previously shown that a short-lived intermediate becomes populated during the acid-induced unfolding of hMb (27,29 14ϩ . By absorption spectroscopy the formation of this state can be observed at 441 nm (27,29).…”

Direct electrochemistry of hemoglobin in PHEA and its catalysis to H2O2

“…When the glass slide coated with Nafion/Hb-PHEA film was immersed in a pH 7.0 buffer, the 412 nm Soret band of Hb appeared again. The reversibility of the change in conformation should be attributed to highly specific and tightly folding conformation of proteins (Sogbein et al, 2000).…”

Protein isolation through impact desolvation of electrosprayed microdroplets (IDEM): Molecular dynamics simulation

“…In the field of biomolecular mass spectrometry, computational studies have been conducted for understanding the protein structure and stability in different mass spectrometric techniques such as electrospray ionization (ESI), matrix-assisted laser desorption (MALDI), MCI, laser spray, and laser-induced liquid beam ionization (LILBID) [12][13][14][15][16]. Electrospray ionization is a common method to transfer large biomolecular ions from solution to the gas phase [17].…”