## 3 Results and Discussion
### HSA Conformational Change Evaluated Using CD and FTIR Measurements  
CD spectroscopy is a very useful and sensitive technique for obtaining information regarding the secondary protein structure. Far-UV CD measurements in the range of 200-260 nm for HSA with and without PDQL were conducted to explore the structural changes of HSA that occurred during the binding process. The CD spectra of the HSA and PDQL-HSA complex exhibited two negative bands in the UV region at 208 and 222 nm, which are characteristic of \(\alpha\)-helix structural units in proteins (Figure 5). The intensities of the negative bands increased with the addition of PDQL without a change in either the position or the shape of the peak. The calculated values for the fractions of the \(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn and random coil structures are listed in Table 8. The results indicated some loss in the \(\alpha\)-helical structures and an increase in the disordered structural content in HSA after the addition of PDQL, in particular isopropyl-modified PDQL, which may be caused by the enhanced binding affinity from isopropyl substitution Therefore, the binding of PDQL may cause secondary structural changes in HSA.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Compound** & \(\Delta\Delta H\) (kJ\(\cdot\)mol\({}^{-1}\)) & \(\Delta\Delta S\) (mol\({}^{-1}\)\(\cdot\)K\({}^{-1}\)) \\ \hline R = CF\({}_{3}\) & \(-\)2.74428 & \(-\)5.1177 \\ R = OCH\({}_{3}\) & \(-\)1.58456 & \(-\)1.1395 \\ R = CH(CH\({}_{3}\))\({}_{2}\) & \(-\)4.78516 & \(-\)8.6412 \\ R = CH\({}_{3}\) & \(-\)4.85363 & \(-\)12.0553 \\ R = CN & \(-\)4.65983 & \(-\)8.9857 \\ \hline \hline \end{tabular}
\end{table}
Table 6: Values of \(\Delta\Delta H\) and \(\Delta\Delta S\).  
To further verify the conformation changes of HSA with PDQL, FTIR spectroscopy was performed. The structural changes of HSA after binding to PDQL were observed using FTIR (Figure 6). Two bands in the secondary structure of a protein were observed by [PERSON] et al. [13], and these bands are the most widely-used vibrational bands in studies of changes in the HSA secondary structure using FTIR spectra in the range of 1700-1500 cm\({}^{-1}\). The amide I band (1700-1600 cm\({}^{-1}\), mainly the C=O stretch) and the amide II band (1600-1500 cm\({}^{-1}\), a C-N stretching coupled with N-H bending) are correlated with the secondary structure of the protein. With the addition of PDQL to HSA, the peak position of amide I changed from 1652 cm\({}^{-1}\) to 1668 cm\({}^{-1}\), and the amide II peak shifted from 1551 to 1547 cm\({}^{-1}\). Furthermore, the intensity of the amide I and amide II bands decreased, and the peak shape changed, revealing that the PDQLs interacted with the C=O and C-N groups in the protein structural subunits. This finding suggested that the PDQLs could also induce conformational changes in HSA during the binding process, as indicated by a comparison with the results of the CD experiments.  
Figure 5: Far-UV circular dichroism (CD) spectra of the PDQL-HSA system: \(C_{\rm HSA}\) = 1.0 \(\times\) 10\({}^{-6}\) M and 1.0 \(\times\) 10\({}^{-6}\) M HSA + 2.0 \(\times\) 10\({}^{-6}\) M PDQL.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multicolumn{4}{c}{**Secondary Structure (\%)**} \\ \cline{2-5}  & \(\alpha\)**-Helix** & \(\beta\)**-Sheet** & \(\beta\)**-Turn** & **Random Coil** \\ \hline HSA & 38.8 & 23.6 & 11.0 & 26.6 \\ HSA + 3a (1:2) & 35.8 & 22.7 & 14.2 & 27.2 \\ HSA + 3b (1:2) & 37.8 & 24.7 & 10.7 & 26.8 \\ HSA + 3c (1:2) & 36.4 & 24.4 & 11.1 & 28.1 \\ HSA + 3d (1:2) & 36.4 & 20.0 & 14.8 & 28.8 \\ HSA + 3e (1:2) & 36.8 & 22.2 & 12.4 & 28.5 \\ HSA + 3f (1:2) & 33.7 & 14.5 & 19.5 & 32.2 \\ \hline \hline \end{tabular}
\end{table}
Table 8: Conformation changes in the secondary structure of HSA with and without PDQL.