Proteins 3-Proton elimination

The racemisation of amino acids bound in proteins is accompanied by -elimination of certain functional groups in side chains of amino acids, which yields dehydroproteins and subsequently cross-links in protein molecules (see Section 2.5.1.3.4). Racemisation begins with a-proton elimination of protein-bound amino acids, giving rise to an immediately isomerisable intermediate car-banion. This carbanion reacts with a proton (hydronium ion), which gives an equimolar mixture of the D-enantiomer and the original L-amino acid bound in protein. Proteolysis then releases the free D-amino acid (Figure 2.39). 

The simplest approach to minimizing protein-wall interaction is to use a buffer pH at which interactions do not occur. At acidic pH the silanols on the surface of the capillary are protonated, and the net charge of the proteins is positive. At high pH, the wall is negatively charged, and so are the sample components. Both conditions result in electrostatic repulsion. Problems associated with operation at pH extremes include the potential instability of proteins (denaturation, degradation, and precipitation) and the limited pH range in which to achieve resolution. Additionally, operation at extreme pH does not eliminate all nonspecific interactions. 

Another way to detect small molecules in the final formulated protein product without the interference from the protein signals is to remove the protein by ultrafiltration. Figure 12.4 compares a section of the proton NMR spectra of a biopharmaceutical protein product before (upper spectrum) and after (bottom spectrum) the protein was removed by ultrafiltering the sample with a Centricon-10 (Millipore Corp, Bedford, MA). Removing protein results in a flatter baseline (bottom spectrum). If small molecules are present in a protein sample, the removal of the protein may allow for unobstructed detection of the small molecules. In this case, a small amount of acetate ( 1 pg/rnl) is detected in the sample [bottom trace, Figure 12.4], Figure 12.5 shows that spikes of 10 p.g/ml of acetate and MES into the protein sample are fully recovered after the ultrafiltration to remove the protein. This example demonstrates that the interference of protein with the detection and quantitation of small-molecule impurities in a formulated protein product can be effectively eliminated by ultrafiltration. 

Clozapine and olanzapine are atypical antipsychotic drugs used in the treatment of schizophrenia. Their strnctnres are depicted in Scheme 2.36. The use of clozapine and olanzapine, which are more effective than standard neuroleptic drugs in the treatment of refractory schizophrenia, is, however, limited becanse of their adverse effects. These adverse effects are ascribed to the formation of the corresponding cation-radicals in living organisms under oxidation by bone marrow cells. These cation-radicals eliminate protons from the NH fragments and generate their nitrenium cations. The nitreninm cations are covalently bonnd to the life-important proteins. This results in the toxic effects of clozapine and olanzapine (Sikora et al. 2007). 

Denaturation. Proteins are quite susceptible to denaturation in alkaline solution because of decreased stabilization of the tertiary structure by elimination of electrostatic interactions between carboxylate and protonated amino and guanidinium groupings (Equations 1 and 2) and hydrogen bonding between the hydroxyl group of tyrosine and carboxylate groups (Equation 3). 

Figure 1. Postulated mechanism of racemization and lysinoalanine formation via a common carbanion intermediate. Note that two B-elimination pathways are possible (a) a concerted, one-step process (A) forming the dehydroprotein directly and (b) a two-step process (B) via a carbanion intermediate. The carbanion, which has lost the original asymmetry, can recombine with a proton to regenerate the original amino acid residue which is now racemic. Proton transfer may take place from the environment of the carbanion or from adjacent NH groups, as illustrated. Protein anions and carbanions can also participate in nucleophilic addition and displacement reactions (24, 82, 83).

Hydroxide ion abstracts an acidic hydrogen atom (proton) from an a-carbon atom of an amino acid residue to form an intermediate carbanion. The carbanion, which has lost the original asymmetry of the amino acid residue, can either recombine with a proton to reform a racemized residue in the original amino acid side chain or undergo the indicated elimination to form a dehydroalanine side chain. The dehydroalanine then combines with an e-amino group of a lysine side chain to form a crosslinked protein which on hydrolysis yields free lysinoalanine (6, 9). 

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