µM Cyt c Red also displayed an additional maximum at 431 nm (Figure ). (Figure ). Cyt c was coupled to the nanoparticles using MUA as a ligand and conjugating with EDC/NHS coupling chemistry, showed less well-defined CD spectra when compared to Cyt c free in solution (Figure and D), although the position of one minimum at 208 nm was maintained (Figure ). Despite this feature, the minima at 222 nm in the free protein were red-shifted to 227 nm, suggesting a change in alpha-helical content and, therefore, an alteration in the secondary structure of the 20, 50 and 100 nm AuNPs. Cyt c was physiosorbed to AuNPs functionalised with MUA via electrostatic interactions presented minima at 208 nm but showed the 222 nm minima to be red-shifted to 225 nm for the 20 nm AuNPs, and to 227 nm for 50 and 100 nm AuNPs, once again suggesting changes in alpha-helical content and subsequently in the secondary structure (Figure ). The definition of the CD far-UV spectra becomes less as the AuNP size deceased for Cyt c conjugated to MUA electrostatically, likely the result of a lower overall content of Cyt c in the samples with larger AuNPs. The CD spectra in the near-UV region showed double minima at 282 nm and 289 nm for all Cyt c conjugated to AuNPs using HS-PEG-COOH (Figure ) and MUA covalently (Figure ), indicating tertiary structure (signified by Tryptophan residues) remained unchanged (Figure and). The and near-UV region, which could account for the difficulty foreseen in determining maxima and minima and be the result of spectral shape changes.