Protonation states usual values

Possible methods of determining the extent of protonation include absorption spectroscopy at a wavelength for which species with n and fir-1 protons have different extinction coefficients, freezing point depression, and electrical conductivity (15). Of these, we have utilized only spectroscopy, which has the disadvantage that only the equilibrium constants for the most highly protonated states are accessible if, as is usual, the species with low protonation are insoluble. In this method, the extinction coefficient c of the compound is determined as a function of the H2SO4 content in the sulfuric acid solvent and correlated with the Hammet acidity function H0 (18) to give the pKB value of the protonated species,... 

The V/K profile is sensitive to the pK values of groups on the substrate or the enzyme form with which it combines that have a required protonation state for binding and/or catalysis. Since catalysis is usually more sensitive to correct protonation state than binding, one tends to see few partial effects in V/K profiles that is, when they begin to drop they keep on decreasing. For nonsticky substrates the profiles have simple shapes, and the pK values seen are the correct ones. Thus, if the physiological substrate is known to be sticky, one should always look for a slower alternate substrate to use for pH studies. 

Assign charges to amino add side chains. The active site residue protonation state is important and can have a significant effect on binding affinity. The protonation states of amino acid residues such as His, Lys, Glu, and Asp are usually based on their pX values but can be influenced by neighboring donor-acceptor groups. 

ESI-MS studies at neutral pH of proteins in their native state usually results in the attachment of far fewer protons compared with ionization from acidified solution. Consequently miz values are frequently 5000 or more and may range up to 10 000. Such species are beyond the normal working range of many mass spectrometers including most quadrupoles and ion traps, but they can be studied with TOE or high field magnetic instruments. 

The steady-state heteronuclear 15N 1H NOEs are determined as a ratio of cross-peak intensities in two experiments, with and without presaturation of amide 1H nuclear spins, usually referred to as NOE and NONOE experiments, respectively. Magnetization exchange between amide protons and water protons could affect the equilibrium 1H magnetization in the NONOE experiment, and thereby increase the measured NOE values. 

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