PLATE

The protein fraction from 70 percent saturation of recrystallised ammonium sulphate was found to have the maximum protein content (19.6 mg/g flower) and hence it was selected for further studies and is abbreviated as PAF in the present study. 4.2.1 Characterization of the selected PAF by Native PAGE and SDS PAGE The selected PAF was characterized by column chromatography. This showed a single peak and was further characterized in native PAGE and SDS page. The results are shown in Plate the SDS-PAGE analysis of the ammonium sulphate precipitated protein extract showed 15 different protein bands with good visibility in CBB R250 staining method (Figure). PLATE CHARACTER IZATION OF PFPa BY PAGE AND SDS PAGE A – Standard Protein Markers; B – PAGE; C– SDS PAGE Each fraction showed a number of major and minor bands indicating several proteins. Of the several bands obtained in 70 per cent saturation of ammonium sulphate, the major band alone was eluted. In order to find out the presence of subunits in this band, it was further subjected to SDS PAGE. The results showed one major protein indicating the absence of subunits. The molecular weight of this protein was found to be 99 KD when compared to the standard molecular markers. 4.3. Fifty percent effective dose of selected protein fraction of Plumeria alba The free radical scavenging capacity of PFC was tested by its ability to bleach the stable DPPH. The DPPH (2,2 diphenyl -1- picryl hydrazyl) radical scavenging activity was carried out using different concentrations of PFC (Figure). The radical scavenging activity was found to be dose dependent. Figure Percentage Free Radical Scavenging Activity of Selected protein fraction of Plumeria alba The protein fraction of Plumeria flower extract showed the dose dependent DPPH radical scavenging activity. From the graph, the 50 percent effective concentrations of these were found to be 28 Â µg and 35 Â µg and used in the further studies. Free radicals and their scavenging systems play important role in the healing of normal and delayed types of wounds. The dose response curve of DPPH radical scavenging activity 0 10 20 30 40 50 60 70 80 90 10010 20 30 40 50 60 70 80 90 100 Ascorbic acid Plant sampleDPPH RadicalScavenging 9(%) of the extract and standards showed that at the highest concentration (0.5mg ml-1) the scavenging effect of the methanolic extract reached 9.3% (Afolayan et al., 2008). Shyuret al. (2005) also reported that the scavenging activity for free radicals of 1,1diphyryl-2-picrylhydrazyl (DPPH) has been widely used to evaluate the antioxidant activity of natural products from plants. The antioxidant activities of the leafy vegetables of India were measured in different systems of assays such as DPPH assay, super oxide radical scavenging assay, hydroxyl radical scavenging assay and lipid peroxidation assay and IC50 values were calculated (Dasgupta and De 2007). 4.4. Matrix-Assisted Laser Desorption/Ionization – Time of Flight Mass Spectra (MALDI-TOF MS) MALDI-TOF mass spectra were used for the analysis of peptide mass fingerprinting and MS/MS ion search identification of the in-gel trypsin digested protein fragments (Figure 5), selected peptide masses were submitted to Mascot (http://www.matrixscience.com) for SwissProt databases search. There was no conclusive match in peptide mass fingerprinting, since MS/MS ion search program was selected for further identification. The MS/MS ion search in the NCBIProt database revealed that, WRKY transcription factor WRKY24-like isoform X1 [Juglans regia] (Figure 6-7) with the protein score of 81 (Protein score is 10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 80 are significant (p