Kutay KARTOZ, Ertuğ AVCI, Mustafa ÇULHA* Department of Genetics and Bioengineering, Yeditepe University, Istanbul 34755, Turkey 10 µM Hemoglobin Melting point:42°C 10 µM Cytochrome c Melting Point:85°C 1 µM Lysozyme Melting Point:75°C RESULTS INVESTIGATION of PROTEIN MELTING PROFILES with SURFACE-ENHANCED RAMAN SCATTERING ABSTRACT INTRODUCTION MATERIALS & METHODS DISCUSSION REFERENCES 60°C 70°C 80°C 30°C 40°C 50°C 90°C Figure 1. Procedure for SERS spectra acquisition. Suspended droplet on CaF 2 surface Protein AgNP Chemicals Hemoglobin, lysozyme, cytochrome c, silver nitrate, and sodium hydroxide were purchased from Sigma- Aldrich (Germany). Hydroxylamine hydrochloride was purchased from Merck (Germany). Methods Silver nanoparticles (AgNPs) (35 nm) were synthesized using hydroxlyamine hydrochloride. Then, 100 µl of proteins were with 100 µl 16 times concentrated AgNP colloid. 2 μl of the mixture was spotted on a CaF 2 slide and dried. A Raman microscopy system (InVia Reflex, Renishaw, UK) was used with laser at 830 nm and 50x objective to perform all SERS measurements. The size distribution and zeta potential measurements of the proteins and nanoparticles were performed using Zetasizer NanoZS (Malvern) at 25°C. Figure 2. SERS spectra of proteins at 30° C and spectral patterns of proteins at different temperatures. In this current study, we used colloidal AgNPs as substrates and the human proteins; hemoglobin, cytochrome c, and lysozyme as models. The proteins were simply mixed with colloidal suspension of AgNPs and dried at suspended position from a hydrophobic surface. We investigated the conformational changes in the structures of the proteins among AgNP aggregates in dried droplet area with SERS at a temperature gradient from 30°C to 90°C. We set the starting temperature to 30°C due to its closeness to the room temperature. The denaturation profiles of the model proteins are found unique and significantly different from each other, which can be used for their identification and detection in protein mixtures. As the end product of gene expression, proteins carry out numerous functions in living systems. Therefore, their detection and identification have critical importance in many fields such as medicine, biotechnology, and proteomics. The mass spectroscopy (MS) and immunoassay-based methods are the most frequently used approaches for the goal [1]. Surface-enhanced Raman scattering (SERS) is a nondestructive vibrational spectroscopic technique that can provide detailed information about the chemical structure of molecules or molecular structures with high sensitivity [2]. Due to the versatility of the technique, SERS has been used for protein detection and identification and its use in this field is increasing day by day [3]. In this study, we investigated protein melting profiles with SERS using three proteins as models. Results are promising and use of the technique can be extended to other proteins and detection of proteins in mixtures. [1] J.Lietal.,“Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer,” Clin. Chem. 48(8), 1296–1304 (2002). [2] S. Lee et al., “Biological imaging of HEK293 cells expressing PLCgamma1 using surface-enhanced Raman microscopy,” Anal.Chem. 79(3), 916–922 (2007). [3] M.Culha and S.Keskin,“Surface-enhanced Raman scattering for label free protein detection and identification,” Proc. SPIE 8234, 823407 (2012). In this study, denaturing temperatures of the proteins were evaluated using SERS. Hemoglobin, cytochrome c, and lysozyme in buffer solutions have melting temperatures at around 42°C, 85°C, and 75°C, respectively. As seen on figure 2, SERS intensity profile of hemoglobin betwen 1400-1600 cm -1 remains almost constant at 50°C. After that temperature, profile changes due to denaturation. Lysozyme melting profiles show remarkable similarity to its melting temperature. Temperatures higher than 70°C, 1400-1600 cm -1 range remains more or less the same. Analysis of the melting profiles of cytochrome c is a bit more complex than the other two proteins due to structural differences among proteins. Interactions of proteins with AgNPs may change their melting temperatures. Nevertheless, changes at 60-70°C demonstrates structural changes as the temperature gets close to the melting temperature.