The molecular mechanism of interaction between SDS and proteins is not clearly understood so far. According to the current knowledge SDS is known to interact with the hydrophobic regions of the proteins. Tryptophan and tyrosine are hydrophobic and hydrophilic aromatic amino acids respectively, which are also known for their intrinsic fluorescence nature in proteins. By observing the autofluorescence of both these hydrophobic and hydrophilic amino acids upon SDS treatment, information about SDS-protein interactions could be obtained. In the present study we have recorded the autofluorescence spectra of five globular proteins [Bovine serum albumin (BSA), Human serum albumin (HSA), Ribonuclease A (RNase A), Lysozyme and Trypsin] by the sequential excitation from 260nm to 295nm at every 5nm intervals. The results obtained clearly indicated BSA and HSA undergone hydrophobic collapse around their tryptophan moieties due to the increased folding of their secondary and tertiary structures upon SDS treatment. Trypsin on the other hand showed complete unfolding upon treatment with SDS. Lysozyme and RNase A did not show any difference in their autofluorescence upon SDS treatment may be due to the stability and fluorophores composition in them. The above results obtained with specific UV excitations clearly shown the tertiary folding and ensembles of the secondary and tertiary structures upon SDS treatment is governed by their stability and bonds stabilizing the proteins.
Proteins are the most diverse and functionally active biomolecules in the living system. In order to understand their diversity and dynamic functionality, visualization in native form without altering structural and functional properties during the separation from the complex mixtures is very much essential. In the present study, a sensitive methodology for optimal visualization of unstained or untagged proteins in native poly-acrylamide gel electrophoresis (N-PAGE) has been developed where, concentration of the acrylamide and bis-acrylamide mixture, Percentage of the gel, fixing of the N-PAGE by methanol: acetic acid: water and washing of the gel in the mili-Q water has been optimized for highest sensitivity using laser induced autofluorescence. The outcome with bovine serum albumin (BSA) in PAGE was found to be highest at acrylamide and bis-acrylamide concentrations of 29.2 and 0.8 respectively in 12% N-PAGE. After the electrophoresis run, washing of the N-PAGE immediately with miliQ water for 12 times and eliminating the methanol: acetic acid: water, fixing of the N-PAGE yielded better sensitivity of visualization. Using the above methodology 25ng of BSA protein band in PAGE was clearly identified by the technique. The currently used staining techniques for the visualization of proteins are coomassie brilliant blue and silver staining, have the sensitivity of 100ng and 5ng respectively. The current methodology was found to be more sensitive as compared to coomassie staining and less sensitive compared to silver staining respectively. The added advantage of this methodology is the faster visualization of proteins without altering their structure and functional properties.
Autofluorescence characteristics of human serum albumin (HSA) are highly sensitive to its local environment. Identification and characterization of the proteins in normal and disease conditions may have great clinical implications. Aim of the present study was to understand how autofluorescence properties of HSA varies with denaturation under urea (3.0M, 6.0M, 9.0M) and guanidine hydrochloride (GnHCl) (2.0M, 4.0M, 6.0M) as well as digestion with trypsin. Towards this, we have recorded the corresponding autofluorescence spectra of HSA at 281nm laser excitation and compared the outcomes. Although, HSA contains 1 tryptophan and 17 tyrosine residues, it has shown intense autofluorescence due to tryptophan as compared to the tyrosine in native form, which may be due to the fluorescence resonance energy transfer (FRET) from tyrosine to tryptophan. As the unfolding progresses in denatured and digested forms of the protein, a clear increase in tyrosine fluorescence as compared to tryptophan was observed, which may be due to the increase of tryptophan - tyrosine separation disturbing the FRET between them resulting in differences in the overall autofluorescence properties. The decrease in tryptophan fluorescence of around 17% in urea denatured, 32% in GnHCl denatured and 96% in tryptic digested HSA was observed as compared to its native form. The obtained results show a clear decrease in FRET between tyrosine and tryptophan residues with the progression of unfolding and urea seems to be less efficient than GnHCl in unfolding of HSA. These results demonstrate the potential of autofluorescence in characterizing proteins in general and HSA in particular.