Kutay KARTOZ, Ertuğ AVCI, Mustafa ÇULHA*Department of Genetics and Bioengineering, Yeditepe University, Istanbul 34755, Turkey

10 µM HemoglobinMelting point:42°C

10 µM Cytochrome cMelting Point:85°C

1 µM LysozymeMelting Point:75°C

RESULTS

INVESTIGATION of PROTEIN MELTING PROFILES withSURFACE-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 CaF2 surfaceProtein AgNP

Chemicals

Hemoglobin, lysozyme, cytochrome c, silver nitrate,and sodium hydroxide were purchased from Sigma-Aldrich (Germany). Hydroxylamine hydrochloride waspurchased from Merck (Germany).

MethodsSilver nanoparticles (AgNPs) (35 nm) were synthesizedusing hydroxlyamine hydrochloride. Then, 100 µl ofproteins were with 100 µl 16 times concentratedAgNP colloid. 2 μl of the mixture was spotted on aCaF2 slide and dried.A Raman microscopy system (InVia Reflex, Renishaw,UK) was used with laser at 830 nm and 50x objectiveto perform all SERS measurements.The size distribution and zeta potential measurementsof the proteins and nanoparticles were performedusing 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 humanproteins; hemoglobin, cytochrome c, and lysozyme as models. Theproteins were simply mixed with colloidal suspension of AgNPs and driedat suspended position from a hydrophobic surface. We investigated theconformational changes in the structures of the proteins among AgNPaggregates in dried droplet area with SERS at a temperature gradient from30°C to 90°C. We set the starting temperature to 30°C due to its closenessto the room temperature. The denaturation profiles of the model proteinsare found unique and significantly different from each other, which can beused for their identification and detection in protein mixtures.

As the end product of gene expression, proteins carry out numerous functions inliving systems. Therefore, their detection and identification have critical importance inmany fields such as medicine, biotechnology, and proteomics. The mass spectroscopy(MS) and immunoassay-based methods are the most frequently used approaches forthe goal [1].Surface-enhanced Raman scattering (SERS) is a nondestructive vibrationalspectroscopic technique that can provide detailed information about the chemicalstructure of molecules or molecular structures with high sensitivity [2].Due to the versatility of the technique, SERS has been used for protein detection andidentification and its use in this field is increasing day by day [3]. In this study, weinvestigated protein melting profiles with SERS using three proteins as models. Resultsare promising and use of the technique can be extended to other proteins anddetection 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 wereevaluated using SERS. Hemoglobin, cytochrome c, andlysozyme in buffer solutions have melting temperatures ataround 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 meltingprofiles show remarkable similarity to its meltingtemperature. Temperatures higher than 70°C, 1400-1600 cm -1

range remains more or less the same. Analysis of the meltingprofiles of cytochrome c is a bit more complex than the othertwo proteins due to structural differences among proteins.Interactions of proteins with AgNPs may change their meltingtemperatures. Nevertheless, changes at 60-70°Cdemonstrates structural changes as the temperature getsclose to the melting temperature.