Dichroism, circular, detection acid residues

It is often difficult to establish that a recombinant protein manufactured in a novel cell system by genetically engineered approaches has the same tertiary structure as the authentic wild-type protein. Because detection by spectroscopic techniques such as circular dichroism (CD) of subtle conformational differences that may arise due to minor perturbations in the hydrophobic regions of a protein is often difficult, the observation of identical retention times for the rDNA and the wild-type protein by RPC and HIC procedures is a useful indicator of common three-dimensional structures. Moreover, when small structural variations arise due to amino acid residue additions, replacement, or chemical/enzymatic modifications, the use of RPC and HIC procedures under less denaturing elution conditions often favors the resolution of these species from the parent protein. The separation of recombinant Met -hGH from recombinant hGH is an example [385,404,415] where such approaches have been successfully applied. 

Other IRRAS applications to peptides and proteins. In addition to the pulmonary surfactant system, a variety of other applications employing IRRAS to study peptide and protein conformation and orientation have appeared. The secondary structure conversion of the amyloid (prion)-protein in the normal form into the abnormal form is the main cause of several human and animal diseases, such as Alzheimer s disease [68]. The secondary structure of the first 40 residues of the amyloid protein was detected by circular dichroism (CD) in aqueous solution and with IRRAS at the interface. A stable /1-sheet-enriched state of the amyloid is formed at the air-water interface, in contrast to the initial bulk solution containing high a-helix/random coil and low /l-sheet parts. The change in the pH going from bulk (alkaline pH) to the interface (neutral or slightly acidic pH) can have effects on the conformation at the interface. Another alternative might be the intrinsic hydrophobicity of the air-water interface, which is a hydrophobic-hydrophilic system with air as the hydrophobic part. 

HSA consists of 585 amino acids and is comparatively rich in cysteine, which allow HSA to form a total of 17 internal disulfide bridges [17]. HSA has a rather high a-helical content [18], which can readily be detected using the circular dichroism (CD) technique. HSA consists of three domains with similar primary structure and is glycosylated at residues Asn-342 and Asp-518. The unfolding of HSA is known to proceed in several incremental steps [19]. 

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