Re: Why an how do certain pH ranges affect the beetroot cell membrane?

Dear Alla,
pH affects membranes by affecting the proteins that make up about 70% of 
most cell membranes (less in inactive cells, such as red blood cells, more 
in others, such as those of mitochondria).  proteins are made of amino-
acids and each amino-acid has a variable number of nitrogen and oxygen 
atoms in it.  These can form HYDROGEN bonds with the many hydrogen atoms 
found in the molecule.  The clever thing is, that the protein folds up to 
ensure that the MAXIMUM number of these hydrogen bonds is made.  
When the pH of a solution changes, the position of some of these hydrogen 
atoms also changes.  This is because amino-acids are AMPHOTERIC, and tend 
to stabilise pH.  Thus, they can lose an H+ ion at the COOH [or 'acid'] 
part of the molecule at higher pHs, or gain an H+ ion at the NH2 
[or 'amino'] end of the molecule at lower pHs.
This, in turn, causes the overall shape of the protein to change with pH.  
This is the reason why most enzymes (which need a precisely-shaped 'ACTIVE 
SITE') can only work well at a certain pH.  Unlike heat, the denaturing of 
a protein by changing pH is (normally) REVERSIBLE.
The dye in beetroot (betalain - see notes below) diffuses out of the cell 
when the membrane proteins are damged.  Changing the pH is just one way of 
doing this - others include the use of heat or organic solvents such as 
ethanol or detergents.

NOTES Follow:
BEETROOT PIGMENTS
-	and membranes –

These pigments are betalain pigments (not, as often thought, 
anthocyanins), which they replace in some organisms.  


They are named after the Beet family of plants (Beta) but are also found 
in fungi (Fly Agaric - the red, spotted one!).  
In petals they presumably attract pollinating insects and may be present 
in seeds/fruits to encourage birds to eat them and so disperse the seeds.

Man has selected for colour in beetroot, both because it is more 
attractive but also because it may well be linked to genes for flavour too.
There is no indication that they have any protective function (e.g. 
against UV light or insect/fungal/viral attack).
Unlike anthocyanins, they are not pH indicators – their colour is stable 
over a wide range of pH.  They are oxidised over time (going brown) and 
this may be prevented by 0.1% ascorbic acid ( = Vit.C); they are sometimes 
used as food colourants.

They are found in the vacuole and thus are used as markers for scientists 
who wish to extract intact vacuoles from plants for research.
To extract the pigment, the membranes must be disrupted.  This can be done 
by heat shock, by detergents or by solvents (e.g. ethanol or acidified 
methanol).  Thin slices have a larger surface area and so leak more 
pigment; freezing the beetroot first bursts the cell membranes and kills 
the cells, thus allowing the pigment to be extracted much more quickly.


Effect of Heat:

	When you heat a beetroot, you disrupt the cell membranes. A 
biological membrane is made of a so-called phospholipid bilayer. These are 
formed because the phospholipids that make it up have a polar "water-
loving" (hydrophyllic) head and a “water-hating” (hydrophobic) tail. The  
tails pack together, exposing only the polar heads to the water. The most 
effective way of doing this is to create two blankets one atop the other, 
with the fatty acid tails towards each other. This is the phospholipid 
bilayer. 
	In a cell they form sacks. One goes all around the cell (the 
plasma membrane), others may form vacuoles (such as the tonoplast). Yet 
others may be like stacks of half empty bags (the endothelial reticulum, 
which is also continuous with the nuclear envelope.  In these lipid seas, 
there will be a number of proteins in various degrees of submersion. Some 
span all the bilayer, thus being exposed on both sides. Others just drift 
on either of its surfaces. Typically, you will find that about 70% of a 
cell membrane is protein. The water around and within the compartments 
formed by the phospholipid bilayers is also crammed with protein (= 
cytoplasm). 



	So what happens when you heat this? When you heat something you 
give it energy. Molecules start to spin and vibrate faster. The water will 
expand too. This will have a disruptive effect on any membrane in its way. 
To make things worse, lipids become more fluid as temperature goes up 
(think of what happens when you heat butter) so the membranes become more 
fragile. 
	Proteins are remarkable machines: they're formed of coiled and 
folded strings of amino-acids, held together by hydrogen bonds and 
disulphide bridges. If you heat them too much, they will untangle and 
break apart (vibrations again). When this happens to the proteins spanning 
a lipid membrane, they will form holes that will destroy the delicate 
structure. Now, any pigments in the innermost compartment will spill out.

Half-life:
The half-life of beetroot pigment is 413 mins at 250C but only 83.5 mins 
at 600C.  These values are doubled in 0.1% ascorbic acid.  Metal ions 
speed up the breakdown – iron is particularly effective.
	They are stable between pH 4.0 and 7.0 – indeed, at high 
temperatures they are most stable in a pH between 4.0 and 5.0 – and most 
fruits and vegetables are acidic!

Effect of organic solvents

	If you want to dissolve lipid-embedded pigments, place a beetroot 
in an organic solvent such as acetone and see what you get. You break down 
the structure between the phospholipids, not the phospholipids themselves 
that much. The proteins on the other hand, are truly destroyed.
	The basic structure of the tonoplast is the same as the plasma 
membrane (as described above). In this respect, they're similar, though 
with a higher proportion of protein in the plasma membrane than in the 
tonoplast. (why?)

PIGMENTS IN PLANTS

1.	The anthocyanins are common plant pigments. They are  water-
soluble glycosides with some or all of the sugar groups removed. The 
colour comes from a positive charge distributed over the chemical ring 
system. The colours of the charged anthocyanin pigments are dependent on 
the pH of the intracellular medium containing these pigments.

2.	Many leaves frequently develop red coloration during development, 
at maturity, and during senescence. Most plants produce anthocyanins 
(usually cyanidin glycosides) as the basis of this colour, but members of 
the Caryophyllales produce nitrogenous pigments, betacyanins. 

3.	The betacyanin pigment of beet roots is normally sequestered in 
the vacuole and, by means of of the beet root. Of course if the beet root 
is cut cells are sliced open and the pigment spills out, but if the 
membrane is altered (phospholipid bilayer + proteins) more subtly leakage 
(diffusion) of betacyanin is induced.
Betalains:  What are betalains?

  Betalains are alkaloid pigments that are found in some families of 
plants belonging to the order Caryophyllales, but in no other plants.     
Little is known about the role of betalains.
Betalains are not found in plants containing anthocyanin pigments –      
structurally they are unrelated
  They have also been found in some fungi e.g. Fly Agaric
  They can be divided into betacyanins and betaxanthins based upon their 
molecular structure. 
 Betacyanins generally appear red to red violet in colour - they absorb in 
the 535-550nm range - hence our choice of filter in the colorimeter)

  Betaxanthins generally appear yellow in colour (absorb in the 475-480nm 
range)
 They cause colour in both flowers, fruits and sometimes vegetative organs
 They are found in the vacuole and they are water-soluble.
  
  Beetroot contains 2 Betacyanins - Betanin and a derivative  

Commercial uses:

Beetroot pigment is used commercially as a food dye.  It changes colour 
when heated so can only be used in ice-cream, sweets and other 
confectionary, but it is both cheap and has no known allergic side-effects.
Beetroot itself, of course, is a common salad ingredient – when cooked, 
vinegar is added to the water to lower the pH.  If you read all the above 
notes, you will see why!


Ó IHW October 2003

ADVICE ON WRITING UP A BEETROOT MEMBRANR PRACTICAL (FOR THE UK!!)
BEETROOT COURSEWORK
- temperature or ethanol concentration -

PLANNING:  

The independent variable is the factor that you control.  Thus you need to 
include full details of how you set about ensuring that the values you 
state are as accurate and reliable as possible.

So:
Temperature – use water-baths and measure the actual temperature
allow enough time for the ‘ingredients’ to reach the indicated 
temperature – state in minutes how long.  NB ‘Room Temperature’ does not 
exist in AS coursework – but you can use a water-bath with tap-water, by 
all means…..
Volume	-  what volumes of liquids are you going to use?  Why?  How are 
you going to measure them? Why?
Beetroot 	- How much are you going to use?  Why?  How are you going 
to cut them?  Why?  Is the beetroot uniform?  How are you going to 
randomise your beetroot disks?  What about genetic / growing / storage 
variations in the beetroot?
Reaction vessels -  What are you going to react the beetroot in?  Why?
Time 	- How long are you going to react the beetroot for?  Why did you 
choose this time?  How are you going to measure it?  How are you going to 
ensure each replicate is the same?
Range	- what range of readings are you going to take?  How did you 
decide on this?  Will it be sufficient for a reliable result?
Concentration -  Range sufficient?  Why chose this?
Control	- This will, of course, be part of the range of results taken, and 
so no separate control will be needed.

The dependent variable is the one you measure.

So:
Absorbance -  What are you going to measure this with?  Why did you decide 
to use a Harris   Digital Colorimeter, set up using filter 5 (yellow-
green, peak absorbance at 550 nm?).  How to you propose to calibrate it?  
What is the volume you use in each cuvette?  How will you measure this?
Replicates - 	How many readings are you going to generate per set of 
results?  Clearly, you realise that the minimum is going to be 5.  How 
many replicates are you intending to use?  Can you combine your results 
with any other groups? Are you going to average them? Why?

DIAGRAM: 
	Have you done one? In pencil?  Labelled?

COLLECTION OF DATA
	Do ensure that you have collected your data on a decent bit of 
paper!  These results need to be included in your final submission (as an 
appendix), as well as the neat, fully titled, word-processed table(s) in 
the ‘Results’ section of your report.  
	Ensure your results are taken to an appropriate degree of accuracy 
and that you have enough in each set (min 5) and enough repetitions (min 
3).  You can pool results with others and average them, without fear, 
providing you state that they are pooled data.
GRAPHING DATA:
	
Titled.  Lines labelled. Axes right way round – independent variable on 
the X axis, and fully labelled -with units!  Most of graph paper area 
used – don’t plot on computer – the exam board don’t like them! Line of 
best fit optional; ‘joining up the dots’ = compulsory!
Take care not to extrapolate back to the origin, unless you are certain 
that that is correct (it usually is not!)
Error bars might be a worthwhile inclusion – if a histogram/bar chart is 
used.
Calculations. As well as a bit of averaging, you might well be able to 
calculate a rate or two – anything (simple!) like this helps to give an 
air of authority to your conclusions.

INTERPRETING

Describe  the pattern of your results – using actual numbers!  This seems 
pointless and even offensive, given that the examiner can read and 
interpret the graph as well as you can.  But do it!  
Conclusions need to be detailed.  This means that you have to give an 
interpretation of your results.
In this case, the purple pigment (probably betanin) is found in the 
vacuole in the centre of the cell.  It is water soluble.  It is surrounded 
by both the vacuole and the cell membranes.  The cellulose cell wall on 
the outside of the cell is fully permeable and so no barrier to the egress 
(= release) of the pigment.
The pigment comes out because the two membranes are damaged.  How?  
Membranes contain c.30% phospholipid (soluble in ethanol) and c.70% 
protein (denatured by high temps).  So your account should explain what 
happens.
The pigment is heat labile i.e. is denatured by heat – turning to a yellow 
chemical.  Since the colorimeter is set to measure absorbance at one 
wavelength, a change in colour will result in a lower absorbance at that 
wavelength and so the numbers decrease.
How does the pigment leave the cell (and why?)?  The concentration outside 
the cell is low – so diffusion will be important.  What factors affect the 
rate of diffusion?  Did you leave the beetroot in the experiment for the 
optimum time?  What would happen with (much) longer and shorter times?

[See handout on Beetroot pigments]

ANALYSING

	The main source of error in any biological experiment is usually 
the natural variation of living things.  What did you do to ensure that 
this variation was minimised?
	The apparatus is more than accurate enough – but what about the 
disks themselves?
	‘Experimental error’ counts for nothing – unless you detail the 
cause of the error and (better) indicate how the experiment could have 
been improved to reduce/eliminate the error that you have identified.
	Clearly, even with pooled class results only a limited range of 
results were abtained; what should be done to ensure that the results were 
reliable, repeatable and applicable to other situations?
	Simply repeating the experiment with the same apparatus and the 
same range of results will improve the reliability but not the accuracy.  
For that, the experiment must be modified – perhaps by using different 
apparatus (which we might not have – such as a more accurate colorimeter, 
which measures light over a much narrower range of wavelengths)
	Your anomalous results (or the class average’s) must be indicated  
on the graph(s) then seek to explain them – i.e. what is the most probable 
cause(s) of these results?  Once again ‘Because that idiot XXXX did it’ 
won’t get any credit at all!

Ó IHW October 2003