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A simple method for extraction and purification of total soluble proteins. Pascale Maillard INRA, UMR Ecologie et Ecophysiologie Forestières, F-54280 Champenoux. Introduction.
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A simple method for extraction and purification of total soluble proteins Pascale Maillard INRA, UMR Ecologie et Ecophysiologie Forestières, F-54280 Champenoux
Introduction Numerous methods exist allowing extraction and purification of total soluble proteins but only some among them can be used in the context of research on stable isotope fractioning or allocation of labelled elements (13C, 15N) between biochemical compounds. The main reasons are the imperative necessity to: • prevent during the experimental procedure contamination of the extract by exogenous carbon • extract each family of proteins (characterized by their solubility in alkaline, aqueous, saline buffers) present in the vegetal material during the same operation
Soluble protein extraction In litterature some solvents are often used to extract total soluble proteins: • SDS/Tris Buffer : SDS (Sodium dodecyl sulfate) 0.069M, tris (tri-hydroxyl amino methane) 0.1M, HCL Ph 8.0 (Bottomley et al., 1982) • Pronase-Tris buffer : (0.4 ml pronase/ml 0.05M tris adjusted with HCL to pH 7.4) (Dickson, 1979, Physiol. Plant.45:480-488; Kleiner et al., 1999, Oecologia 119: 408-418) • Sodium Phosphate buffer : (different molarities are used 10 to 100mM) Ph range: 6.8 to 7.8 (Deléens et al., Planta, 1977; Cliquet et al., 1990, Plant Physiology) • The buffer solution can be a source of contamination by the carbon it contains, the last one is therefore used.
First step: extraction • 200 to 300 mg of lyophilized vegetal powder are homogeneized in 50 mM Na phosphate buffer (pH 7.4). 20% of PVPP is added to trap phenol compounds For ex.: for 100 ml buffer to 80, 5 ml KH2PO4 (50mM) add 19.5 ml Na2HPO4 (50 mM) • The homogenate can be (1) gently mixed at room temperature during a night or (2) ground during several minutes in a mortar to optimize the extraction in the buffer • The homogenate is centrifugated (12 000 G 10 mn), The pellet is assumed to contain the insoluble protein fraction. The supernatant contains the soluble protein fraction.
Second step: purification • Supernatant containing soluble compounds is kept and submitted to heat denaturation (60 mn 100°C). • A drop of HCl 6 N (or TCA) is added (to avoid a possible renaturation of some soluble proteins). • Storage during a night in a refrigerator to optimize the precipitation of denatured proteins • Centrifugation 12000 G 10 mn. • The pellet contained denatured proteins only • This pellet is washed two times with 5 ml ultra pure water to eliminate the buffer salts then dried under vacuum • The total dried residue is weighed before mass spectrometry analysis. giving information on the recovering of the compound
First Problems encountered: • For the moment this method was assayed only on inner bark of pine trees • Surprisingly, N concentration is low compared (50-60% C and less than 1% of N, d13C=-27 to -29 per mille) • Why?: degradation of soluble proteins,loss during washing, contamination by other compounds, low N is due to the particular nature of proteins in wood (glycoproteins?)
Comments of a beginner ??? Each tissue, a fortiori each species, have to be treated as a particular case due to intrinsic variability in biochemical compounds Finally, to be confident in this method it is necessary to have informations about: • The quantity, C and N%, delta of soluble proteins in vegetal tissues studied and the percentage of recovering after purification. • The percentage of nitrogen (depending on the nature of amino acids in proteins). Initial content and composition in tissue Black box Final content and composition of the residue
Control of the quality of purified extracts A first control of quality (no alteration of % of C and N and delta’s) and yield is possible needing no sophisticated analytical systems in the laboratory: • - by using a standard (BSA for example), alone or mixed with vegetal sample and comparing C and N isotopic values of BSA before and after extraction • -by a colorimetric measurement of soluble protein concentration in vegetal tissues before and after their purification (Biorad, Bradford method using Coomassie blue) • -comparison with other techniques (ex. pronase tris buffer, with filtration of resulting amino-acids before mass spectrometric analyses More….HPLC, GC-IRMS analyses for identifying amino-acids constituting purified soluble proteins, electrophoresis...
