## 3 Results  
### Characteristics of Obtained L-Asparaginase Conjugates  
In this study, we obtained three conjugates of L-asparaginase RrA with polyamines PEI, PEI-PEG, and spermine using Woodward's reagent. The reactions' schemes are shown in Figure 1. Table 1 illustrates the characteristics of the polyamines used. The obtained conjugates were characterized by gel filtration, CD, and IR spectrometry. Figure 2 shows the chromatograms of the RrA solutions and the RrA-PEI-PEG and RrA-Spm conjugates. The protein fractions were detected at 280 nm. The retention times (t\({}_{\text{R}}\)) corresponding to the major peaks on the chromatogram of the native RrA and its modified forms are shown in Table 2. The native enzyme has a main peak at approximately 24 min and a small peak at 18 min. These two peaks correspond to dimeric and tetrameric forms (36 and 72 MM), which follows a comparison with the control sample, BSA, which has an MW of about 66.5 kDa. Thus, RrA is represented in tetrameric and dimeric forms in the solution. As we can see, the dimeric form is predominant (24 min). The conjugation of RrA with PEG-PEI shifts the equilibrium between the two forms toward the tetrameric form. It is confirmed from the ratio of peaks on the conjugate chromatogram, where the ratio of the peaks at t\({}_{\text{R}}\) 17.6 and 23.9 min is approximately 1:1. The free PEI-PEG copolymer has two small peaks at 24.7 and 26.7 min. The conjugation of RrA with spermine leads to a shift in the equilibrium toward the tetrameric form. A single peak corresponding to the tetrameric form is observed in the RrA-Spm chromatogram. \begin{table}
\begin{tabular}{c c c c c} \hline
**Polymer** & **MM, Da** & **Origin** & **Structure** & **Charge** \\ \hline Polyethyleneimine & 2000 & Synthetic & Linear & Polycation \\ PEI-PEG & 30,000 & Synthetic & Branched & Polycation \\ Spermine & 202 & Natural & Linear & Polycation \\ \hline \end{tabular}
\end{table} 
Table 1: The characteristics of the polyamines used in this work. Figure 1: The reaction scheme of RrA with Woodward’s reagent, polyamines, and resulting conjugates with polyamines. Figure 3 shows the IR spectra of the purified solutions of the RrA-PEI-PEG, RrA-Spm, and RrA-PEI conjugates. The spectra of the unmodified enzyme, as well as the covalent conjugate of RrA with the PEI-PEG copolymer, show characteristic peaks of amide I at 1500-1600 cm\({}^{-1}\) and amide II at 1600-1700 cm\({}^{-1}\), corresponding to the absorption of the amide bonds of the proteins. Additionally, the conjugate has absorption bands at 1460 cm\({}^{-1}\) (N-H vibrations of the secondary amino groups and CH\({}_{2}\) bonds) and 1070 cm\({}^{-1}\) (C-O-C bond vibrations), characteristic of the PEI-PEG copolymer (Figure 3a). The RrA-Spm conjugate also has bands in the amide I and amide II regions (Figure 3b). It has peaks in the 1035-1230 cm\({}^{-1}\) region, corresponding to the C-N bond vibrations in the polyamine molecule. In addition, some contribution to the intensity of the amide I peak can be made by the band at 1620 cm\({}^{-1}\), corresponding to the N-H bond vibrations of the amines. RrA-PEI (Figure 3c) has an intense, broad band at 1050-1150 cm\({}^{-1}\). A band at 1450-1640 cm\({}^{-1}\) (N-H, CH\({}_{2}\)) can also be distinguished in both the polymer and the conjugate. The obtained conjugates were analyzed using CD spectrometry for the content of the secondary structures in comparison with the native enzyme. Figure 4 shows the CD spectra of the native enzyme and the conjugates with PEI, PEI-PEG, and Spm. The shape of the spectrum characteristic of the native enzyme is preserved for the conjugates, as well. The content of the secondary structures changes slightly: in the conjugates, the number of alpha-helices decreases, and the percentage of disordered structures increases (Table 3). The percentage of antiparallel beta structures, which are responsible for aggregation, increases [17].