Amino acid side chains ionizable

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

For amino acid analysis the labeled protein needs to be hydrolyzed and derivatized. Most commonly the hydrolysis is performed in 6 M HC1, and the amino acids are converted into their isopropyl ester and pentafluoropropanamide derivatives (Fig. 1.1) before GC/ MS analysis. The molecular ion is not always visible after standard electron impact (El) ionization, and the fragment after loss of the carboisopropoxy group is the highest observable peak. This leaves m/e=175 plus the mass of the amino acid side chain, from which the degree of labeling can be directly deduced. 

The ionization properties of side-chain substituents will usually carry through into the peptide or protein and influence the behaviour of the polymer. However, the actual pATa values of the amino acid side-chains in the protein are modified somewhat by the position of the amino acid in the chain, and the environment created by other substituents. Typical pATa values are shown in Table 13.2. 

Enzymes are active over a limited pH range the pH value of maximum activity is known as the pH optimum, and this is characteristic of the enzyme. It typically reflects the pH necessary to achieve the appropriate ionization of amino acid side-chains at the active site. 

Since peptides are amphoteric, Zt and Zc are expected to show nonlinear dependencies on pH. Similar behavior has been observed for various synthetic peptides separated on both strong anion and strong cation HP-IEX sorbents. As a consequence, the minima in the In /t iex i versus pH plots at a defined concentration of displacing salt will not usually occur at the predicted p/ value of the peptide, but rather at another pH value. Implementation of an optimized HP-IEX separation of peptides thus requires that the sequence microlocality and extent of ionization of the surface-accessible amino acid side chains, or the N- and C-terminal amino and carboxy groups, respectively, are taken into account. 

The charge properties of amino acids are very important in determining the reactivity of certain amino acid side chains and in the properties they confer on proteins. The charge properties of amino acids in aqueous solution may best be considered under the general treatment of acid-base ionization theory. We find this treatment useful at other points in the text as well. 

An additional point should be noted from table 3.3. Whereas the amino acid side chains (R groups) that are normally charged at physiological pH are restricted to five amino acids (aspartic acid, glutamic acid, lysine, arginine, and sometimes histidine), a number of potentially ionizable R groups are part of other amino acids. These include cysteine, serine, threonine, and tyrosine. The ionization reac-... 

In current structural. models of the Na+ channel, the four homologous a-helix domains pack together around a central pore formed by the four copies of the amphipathic helix 3. These helices probably are oriented so that most of their ionizable amino acid side chains face the aqueous space in the pore, whereas most of their nonpolar side chains face outward and interact with hydrophobic residues of other helices (fig. SI.7). The diameter of such a pore is... 

The enzyme catalyzes the cleavage of the bond between carbon 1 of residue [4] and the oxygen atom of the glycosidic linkage of residue [5]. Two amino acid side chains in the region of this bond can serve as proton donors or acceptors Asp 52 and Glu 35, each of which is about 0.3 nm from the bond. Asp 52 is in a polar environment and is ionized at the pH optimum of lysozyme (pH 5), whereas Glu 35 is in a nonpolar region and is not ionized. The proposed catalytic mechanism is given in Fig. 8-3. 

To describe completely the effects of pH changes on enzyme catalysis is an almost impossible task. Many of the amino acid side chains in an enzyme are ionizable, but in environments with polarities different from that of the free solution, their pKa s (Chap. 3) will probably be significantly altered. However, experimentally, it is a simple matter to determine values of steady-state kinetic parameters (Km, Kmax) of an enzyme for various pH conditions. 

Yuan, M., Namikoshi, M., Otsuki, A., and Sivonen, K. 1998. Effect of amino acid side-chain on fragmentation of cyclic peptide ions differences of electrospray ionization collision-induced decomposition mass spectra of toxic heptapeptide microcystins containing ADM Adda instead of Adda. EurMass Spectrom 4 287-298. 

The perturbation of the acid dissociation constant of an amino acid residue as a consequence of its environment within a protein represents another mechanism for enhancing its reactivity relative to a free amino acid in solution. Most amino acid residues react with their respective modification reagents in their unprotonated form instead of in their conjugate acid form. Eq. (4.10) describes the pH dependence of the simple bimolecular reaction (eq. 4.9) of the free base form of nucleophilic amino acid side chain with a non-ionizable modification reagent where is the acid dissociation constant and Aj is the total concentration of amino acid. 

The rate of enzyme-catalyzed reactions typically shows a marked dependence on pH (Figure 8-7). Many of the enzymes in blood plasma show maximum activity in vitro in the pH range from 7 to 8. However, activity has been observed at pH values as low as 1.5 (pepsin) and as high as 10.5 (ALP). The optimal pH for a given forward reaction may be different from the optimal pH found for the corresponding reverse reaction. The form of tlie pH-dependence curve is a result of a number of separate effects including the ionization of the substrate and the extent of dissociation of certain key amino acid side chains in the protein molecule, both at the active center and elsewhere in the molecule. Both pH and ionic environment will also have an effect on the three-dimensional conformation of the protein and... 

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