title: Laser induced autofluorescence in the monitoring of β-mercaptoethanol mediated photo induced proton coupled electron transfer in proteins.
abstract: Photo induced proton coupled electron transfer (PCET) is an important process that many organisms use for progression of catalytic reactions leading to energy conversion. In the present study, the influence of SDS and BME on the redox properties of tyrosine and tryptophan for five different globular proteins, BSA, HSA, RNase-A, trypsin and lysozyme were studied using laser induced autofluorescence. The proteins were subjected to denaturation under SDS, SDS plus heat and SDS plus β-mercaptoethanol (BME) plus heat and the corresponding fluorescence were recorded. The influence of BME on the autofluorescence properties of the proteins were evaluated upon tris-2-corboxy-ethyl phosphine (TCEP) denaturation. The BSA and HSA when exposed to SDS alone, exhibited hydrophobic collapse around their tryptophan moieties. However, these proteins when treated with SDS plus BME plus heat, an unusual red shift in the emission was observed, may be due to proton transfer from hydroxyl group of the excited tyrosine residues to the local microenvironments. The observation was further confirmed with similar proton transfer in absence of tryptophan in RNase-A showing involvement of tyrosine in the process. A drastic quenching of fluorescence in all of the proteins under study were also observed, may be due to photo-induced electron transfer (PET) from BME to the intrinsic fluorophores resulting in radical ions formation, evaluated upon DCFDA measurements.
authors.affiliation: Department of Biophysics, School of Life Sciences, Manipal University, Manipal, India.
authors.forename: S
authors.initials: S
authors.lastname: Manjunath
authors.affiliation: Department of Radiation Biology and Toxicology, School of Life Sciences, Manipal University, Manipal, India.
authors.forename: B S
authors.initials: BS
authors.lastname: Satish Rao
authors.affiliation: School of Life Sciences, Manipal University, Manipal 576104, Karnataka, India.
authors.forename: K
authors.initials: K
authors.lastname: Satyamoorthy
authors.affiliation: Department of Biophysics, School of Life Sciences, Manipal University, Manipal, India. Electronic address: mahato.kk@manipal.edu.
authors.forename: K K
authors.initials: KK
authors.lastname: Mahato
mesh_terms: D000818:Animals; D002417:Cattle; D004583:Electrons; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D007834:Lasers; D008623:Mercaptoethanol / Q000737:chemistry*; D010720:Phosphines / Q000737:chemistry; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D005452:Fluoresceins; D008623:Mercaptoethanol; D010720:Phosphines; D011506:Proteins; D011522:Protons; D012967:Sodium Dodecyl Sulfate; C029569:diacetyldichlorofluorescein; C080938:tris(2-carboxyethyl)phosphine
keywords: Betamercapto ethanol (BME); Fluorescence emission-shift; Photo-induced electron transfer (PET); Sodium dodecyl sulfate (SDS); Tris-2-corboxy-ethyl phosphine (TCEP)