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In the experiment to show lipid and protein content of membrane what conc and type of acid do you recommend for de-naturing the protein? I have looked up a few old references on plant physiology. In Scottish courses, we tend to use the effect of temperature and the denaturing of the protein in the membrane. Hydrogen ion concentration does however also effect permeability but I'm not convinced that the cause of that effect is simply protein damage but more subtle effects on the three dimensional structures of proteins within the membrane through interference with hydrogen bonding etc. Anyway, whatever the precise mechanism - exodiffusion of pigment from beetroot tissue discs will occur in 0.1 M hydrochloric acid. It will also occur in 0.1 sodium hydroxide when the red pigments won't be (red that is)- they'll be colourless. There will be a yellow colouration though because of the flavenoids which should also leak out. To demonstrate the effects of dissolving out lipid, beetroot discs will also 'leak' in ethanol (IMS) and in detergent solutions. Hypotonic sodium chloride will also do the trick and there's also a simple demo of the effect of ionic balance (antagonism it used to be called) if investigations are done with sodium chloride on its own and then in mixtures with say calcium chloride (keeping the overall ionic concentration the same). Another one is to rapidly freeze and then thaw discs when ice crystal damage will cause exodiffusion. A slightly more complex variant is to subject potato tissue to atmospheres of ethanol (IMS) fumes when the leakage across membranes will bring catchecol oxidase into contact with its substrate(s) and cause the development of first reddish brown and then black polyphenolics. This will be far more rapid in potato discs in closed containers with an exposed sample of ethanol than it would be simply in air with an open dish of water (we used to use chloroform or carbon tetrachloride vapours but that's not on these days!). Another great demo we used to do but I've unfortunately lost my old notes detailing the protocol was to put entire grains of 'Blue barley' (barley with a blue pigment in the aleurone layer) in sulphuric acid. The seed coat is differentially permeable and keeps the acid out. Should you nick the seed coat with a scalpel, however, the acid now penetrates the cell membranes and turns the pigment in the aleurone layer from blue to red. It's an elegant demonstration. A similar demo was done with the house plant Rhoeo spathacea Stearn syns Rhoeo discolor Hance, Tradescantia spathacea Sw. [Boat Lily, Moses-in-the-Cradle, Moses-in-a-Boat]. This has an intracellular (vacuolar I think) pigment which is pH sensitive. If epidermal strips are placed in differing concentrations of acid the colour change can be observed directly at the point when the cell membranes break down and the acid is admitted. As I say, a search has failed to turn up the old note books with the detailed protocols but there should be enough here as bases for project starters. John Richardson
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