ИСТИНА

Angiotensin I-converting enzyme (ACE) is a glycoprotein, consisting of two homologous domains within a single polypeptide chain. The exact 3D-structure of the two-domain ACE is still unknown. ACE produced in different cells is differently glycosylated which may alter some enzyme characteristics. ACE concentration in the blood is an important parameter of clinical observation, its increase or decrease may accompany various pathologies. Of special interest, however, could be the possibility to distinguish ACE in the blood coming from different organs in certain diseases, even in small amounts. Application of surface-enhanced Raman spectroscopy (SERS) provides the qualitative and quantitative analysis of many compounds, including proteins. This is why we tried to provide a method for defining very small amounts of ACE by its SERS spectra. For this purpose, we used special nanostructured matrices based on thin silver films obtained by electron beam evaporation in a vacuum. For the first time, stable SERS spectra of native and thermo-denatured ACE from seminal fluid were obtained and their complete interpretation was carried out. The measurements were carried out at excitation laser wavelength 785 nm, accumulation time of one spectrum 60 sec, and laser power 64 mW. The reproducibility of the spectra was checked by assessing the value of the roof-mean-square deviation over the entire spectrum. Putative ACE degradation during long-term measurements of SERS spectra on the matrices was prevented due to a certain ratio of the root-mean-square roughness of the silver film and its thickness. Based on SERS spectra of native ACE, we characterized the region on the ACE molecule in contact with the silver matrix and demonstrated the model of the two-domain ACE. It appears the C-domain is located in such a way that its long axis is parallel to the silver matrix and the N-domain is almost perpendicular to the long axis of the C-domain. It was found that ACE denaturation led to a significant increase of SERS spectra intensity and to bands shifting. Deglycosylation of denatured ACE resulted in SERS spectra change. The results demonstrate the opportunity to define ACE SERS spectra of native and denatured enzyme forms, as well as deglycosylated ACE, which could form the base for the detection of tissue-specific ACE. Thus, we took a step towards the development of an interdisciplinary direction using SERS on functional nanoplasmonic materials for accurate detection of the enzyme for the purpose of further medical diagnostic methods.