We did not find any clear signature of Asp and His (histidine), two other amino residues of lysozyme, in the SERS spectra of the molecule. 

## C. Disulfide bond 

There are four pairs of S-S bond in lysozyme. Among these four pairs, two pairs of S-S bonds are near the α -helix region and two others are in the β -sheet region. The strong SERS spectrum for vibration of S-S bond appears at 505 cm − 1 just after addition of lysozyme in the Ag sol. However, the intensity of this band decreases with time (Fig. 5(d)). To check the effect of the reducing environment on the disulfide bond, an additional experiment with lysozyme and NaBH 4 was conducted. The trend in the change of relative intensity of S-S bond (Fig. 5(e)) indicates that reduction of the disulphide linkage by BH − 4 (note that the Ag colloidal particles are stabilized by BH − 4 in NaBH 4 stabilized sol) is possible. 

## Discussion 

Here we report the specific amino acid residues of lysozyme molecule which are adsorbed on Ag colloids. Appearance of strong vibrational modes of Trp and Phe residues in the SERS spectra of lysozyme molecule and the similar trend in the change in relative intensities of these modes (Fig. 5(a) and Fig. 5(b)) signify that both these residues are close to the Ag surface and occupying adjacent sites in the adsorbed molecule. A lysozyme molecule contains six Trp residues (Fig. 6). The accessible surface area (ASA) available for these residues are shown in Table II. Three (Trp-62, Trp-63 and Trp-123) of the six Trp residues of a free lysozyme molecule have ASA more than 50 ˚ A 2 and, hence, are likely to be free to bind with a substrate. Trp-62 and Trp-63 are exposed at the edge of the active site cleft and known to interact specifically with a substrate [29]. Trp-123 is close to the c-end of the amino acid chain of the molecule. Trp-28, -108, -111 have relatively less ASA and are expected to be burried. Of all three Phe residues (Phe-3, Phe-34 and Phe-38) of lysozyme molecule the ring of Phe-34 is very close to Trp-123 and its ASA is relatively large compared to that of the other two [Fig. 6 and Table II]. Thus, it is most likely that the lysozyme molecules are adsorbed on Ag surface via the red marked region which includes both Trp-123 and Phe-34 residues of the molecule [Fig. 6]. 

Next we discuss the observed SERS spectra of amide I band. It is to be noted that the ratio of the secondary structure contents i.e α helix: β sheet:random coil in free lysozyme is expected to be 0.54:0.15:1 [32]. Dominant spectral features of α -helical component in Fig. 4 indicate that lysozyme is adsorbed on the colloidal Ag surface either in an α -helical region 

