jybai
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Function of (HPO3)n
Please tell me what is the function of HPO3 for the analysis of the activity of enzyme ,such as SDH or GSH-px.thank you.
[Edited on 28-5-2004 by chemoleo]
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jybai
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function of HPO3
please help me to explain this question that why should we must have the protein precipitation before we analyse or measure the activity of enzyme.
sos,help me?
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vulture
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Don't demand answers and don't double post!
One shouldn't accept or resort to the mutilation of science to appease the mentally impaired.
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chemoleo
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Hmm, what is it with the Biochemistry section that people ask badly-worded questions? 
First of all, what is HPO3? your personal version of phosphoric acid H3PO4? 
In your first question, you ask what 'HPO3' does. Then in the second question you seem to answer yourself, in that it precipitates
proteins?!?!?
Anyway, to my knowledge, TCA (trichloroacetic acid) is generally used to precipitate proteins. It has the disadvantage though that it denatures them,
thus it's unlikely you will be able to measure any activity. Again, please explain what HPO3 is.
In any case, why would you want to precipitate protein, in order to determine its activity? Well, of course to get rid of other contaminants (whcih
remain in solution), and, if you have a supremely superb scale, to determine the amount of protein by weight. This can then be used to estimate the
activity per mg of protein. This does NOT necessarily relate to the molar activity, as your protein preparation may not be pure.
Does that answer your question? If not, then please rephrase it, and explain precisely your problem, rather than just throwing out unexplained details
here and there! Thanks.
[Edited on 28-5-2004 by chemoleo]
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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vulture
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(HPO3)n are polyphosphoric acid of various forms.
Römpp claims they can precipitate proteins..
One shouldn't accept or resort to the mutilation of science to appease the mentally impaired.
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chemoleo
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Ah, I see  . With the bracket and 'n' it makes a lot more sense.
Nonetheless - as it is an acid, most proteins will denature except a few acid resistant ones.
So I guess the only way this question would make sense is to first measure the activity, and then to precipitate the protein to determine the
weight, or measure it by some other means. I dont think this will be immensely accurate, as people normally work with small amounts, i.e. micro or
nanomoles, or less.
Much better would be to measure the absorbance (if the protein is pure), and deduce the concentration via its extinction coefficient. Or, use assays
such as the coomassie one, or BSA etc.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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jybai
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Thank you anyway. Now I am going to measure the activity of SDH and LDH inside the lynphocytes. I have found some data . they detail that before
measuring the activity of enzyme，we need do something. First ，break the cell membrane with Triton –x 100，second 10μl of
cell lysates is mixed with 40μl 10% of HPO3（that is metaphosphoric acid）and then centrifuged for 10 min at 10000rpm.，Third
measure the activity of enzyme such as SDH or LDH or GSH-Px.. I just don’t know why should we use 10% of HPO3 before measuring（some one told
me that is for precipitation of protein）. I wonder will not the SDH or GSH-Px be precipitated together with other protein. Sorry my English is
poor，but I am anxious to get your kind answer. Thank you.
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vulture
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(HPO3)n is going to be a very weak acid IMHO.
One shouldn't accept or resort to the mutilation of science to appease the mentally impaired.
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PHILOU Zrealone
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Yes indeed weak acidity because it corresponds to pKa3 of H3PO4.
Polyphosphoric acid cristalls and polyphosphates are used in some cation exchanger to soften water.
Of course precipitation will occure for the three proteins...the main factor of precipitation is the MM of the protein.
10% is firts not to strong to preserve protein integrity/fuctionality and it will allow common ion effect to help precipitation (it is in excess) and
finally it will increase density of the media sothat most crap will float...but the precipitated protein will sink.
PH Z (PHILOU Zrealone)
"Physic is all what never works; Chemistry is all what stinks and explodes!"-"Life that deadly disease, sexually transmitted."(W.Allen)
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chemoleo
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Yes I guess the acidity won't be a huge problem after all - except for some sensitive proteins. With one of the proteins I work with, for
example, I know it unfolds at a pH below 4.5, and loses its binding activity. Similarly it does so above 55 deg C. I would check the buffered (HPO3)x
solution for its pH, and confirm that your protein in question does not unfold. Just in case you get less activity than expected, or none at all.
Anyway, another precipitant, one that was used for decades, and is still used now and then, is ammonium sulphate. It precipitates proteins, but not
fats etc. Plus it does not alter the pH too much (although I never checked), most protein retain their functionality.
Ammonium sulphate prec. is normally done incrementally, so that the various proteins are precipitated incrementally, too. However, there is no
correlation betw. MW and A.S. concentration, as the precipitation effect depends predominantly on surface charges etc.
During A.S. precipiation, each fraction is taken and centrifuged, with increasing amounts of A.S. The precipitate is white, and some of the fractions
contain your protein. This way you also removed a large fraction of other proteins.
Once you identified which fraction contains your protein, it is redissolved in buffer and dialysed against your assay buffer. Also make sure that your
lysates/protein isolates contain protease inhibitor cocktails, so that none of your protein is proteolytically degraded (unless of course your protein
is pure)
Why would you want to do the precipitation before you measure the activity?
Well, there is plenty of other things in your cell lysate (after triton/detergent disruption of cells), which could interfere with whatever assay you
use to determine the enzyme's activity. Ideally (and this is what you *should* do if you want to be scientifically astute), it should be purified
as far as possible, without losing any of the starting amounts.
[Edited on 27-5-2004 by chemoleo]
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Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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vulture
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Ammoniumsulfate is quite acidic, certainly more acidic than (HPO3)n if that corresponds to the pka3 of H3PO4 like philou said.
One shouldn't accept or resort to the mutilation of science to appease the mentally impaired.
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chemoleo
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Darn... I feared someone might point this out ... and this someone comes by the name of vulturius 
I never heard anything about A.S. denaturing proteins, but I guess one can't outrule that.
All that I tried to point out was that there are precipitating alternatives, without the 'acid' word at the end 
I rest my case.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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PHILOU Zrealone
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I really think that proteins are such weird objects with such a high specificity and several "structure deifinition" (*) that they almost
have specific isolation techniques (like alkaloids) and no general rules may be applied.
(*) Primary, secondary, ternary and quaternary Structure that defines:
-aa sequence
-the position of interacting groups
-the position of bending aa
-type of peptidic folding Beta sheets or alfa helix and their position.
All those parameters are influenced by...
-the polarity and nature of the surrounding solvant
-the temperature
-the pH
-inhibitors, competitors and substrats...via complexation or so.
So following this it is understandable that such large and sensitive molecules are very special and demands a special treatment...that is sometimes
discovered after years of studies...
(NH4)2SO4 will increase the polarity to such an extend that it might be "lethal" to the folding of some proteins...imagine a porine with all
its polar groups inside and hydrophobic groups outside...it will explode and revert as much as possible the hydrophobicity to the core and the
hydrophilicity to the external world.
Many computational studies show that solvants plays a very strong role in the shape of proteins...and as we all know shape defines activity or
desactivation....
Besides that as Vulture said NH4(+) is quite acidic...it easily reach pH 4.5 in solution and here you must have a lot to start precipitation....
SO PLEASE NO GENERALISATION RECIPE!
IT IS 75% of the case WRONG.
PH Z (PHILOU Zrealone)
"Physic is all what never works; Chemistry is all what stinks and explodes!"-"Life that deadly disease, sexually transmitted."(W.Allen)
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Esplosivo
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Besides that as Vulture said NH4(+) is quite acidic...it easily reach pH 4.5 in solution and here you must have a lot to start precipitation....
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Well actually I think that it is the SO4(2-) which gives the acidic nature of ammonium sulphate, not the NH4(+). The ammonium sulphate breaks into
ions and the NH4(+) ion is a weaker base than the SO4(2-) and therefore the solution will be somewhat acidic.
Theory guides, experiment decides.
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PHILOU Zrealone
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Live with than man, SO4(2-) is almost neutral in solution and as a proof... changing NH4(+) for Na(+) change stronly the pH but stil the anion is the
same.
Following the rules of acidity...
NH3 is a moderately strong base
--> its conjugated acid NH4(+) is a weak acid
HSO4(-) is a strong acid
--> its conjugated base SO4(2-) is a very weak base (under the spontaneous hydroxydolyse of water = protolyse of water)...
NaOH is a very strong base
--> Na(+) its conjugated acid is a very weak one
As a reminder:
pKa +pKb = 14 = pH + pOH = 7 + 7
So adding Na(+) to water doesn't do shit; so does adding SO4(2-) but NH4(+) concentration provides you a pH value of approx pH = pKa/2 = 9.25/2
= 4.63
This explains why NH4NO3 is able to dissolve some metal oxydes and hydroxydes (Li2O, CuO, ...)
Since NH4(+) is a stronger acid than Li(+) or Cu(2+) and OH(-) is a stronger base than NO3(-) --> the stronger acid and base react to form a new
compound and the rest rearranges.
NH4NO3 + LiOH --> NH4OH + LiNO3
weakAc + strong B --> weak B + neutral
A neutralisation has occured thus reducing both acidity and basicity of the reactants in the final media.
PH Z (PHILOU Zrealone)
"Physic is all what never works; Chemistry is all what stinks and explodes!"-"Life that deadly disease, sexually transmitted."(W.Allen)
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PHILOU Zrealone
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You stated it right but you mixed all wrong in your conclusion....
NH4(+) is a weaker base than SO4(-)....
Extend this further and enlarge your vison.....and extrapolate
H(+) is an even weaker base than SO4(-) because H(+) is one of the strongest acid.
SO4(-) is a much weaker base than OH(-) which is one of the strongest base.
Thus you have:
H(+) << NH4(+) < SO4(2-) << OH(-) on the basicity scale....
Hence on the acidity scale it is:
H(+) >> NH4(+) > SO4(2-) >> OH(-)
As a conclusion your conclusion was wrong. Because following the very same logic you would aswel proclaim HO(-) to be the reason of acidity character
and H(+) to be inert.
TADAAAAAAHHHHHHHHH
Per absurde demonstrandus
PH Z (PHILOU Zrealone)
"Physic is all what never works; Chemistry is all what stinks and explodes!"-"Life that deadly disease, sexually transmitted."(W.Allen)
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chemoleo
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NO NEED TO SHOUT!
Shouting makes noone 'righter' or 'wronger', 'better' or 'worse' or 'good' and 'evil'. And
it doesn't exactly add to peace, tranquility and friendliness to the forum (the latter being an attribute I particularly value here) .... and indeed, I have a fine set of ears with tiny hairs that are able to pick up
even the most timid noises !
Regarding purification techniques - noone to this point argued (much rather to the contrary) that they can be universally used, for every protein on
earth. Besides this, A.S. precipitation is an old technique, and not much used anymore, precisely for the reason that there are better
techniques, or techniques that can be generalised more often.
Take affinity tags, for example - you link a protein such as GST, MBP, etc or a His Tag to the N-terminus of the protien to be purified, and purify it
(to a relatively high purity) by passing it over a resin to which the affinity protien binds. This way you get rid of >90% impurities. Guess what-
from personal experience, I would say that this technique is successful in MORE than 2/3 of all cases. Except of course in the case of membrane
proteins, and the like. There we go, we have a technique that works well, so you could call that a 'generalised recipe' for
purification of globular soluble proteins, or something along those lines.
Equally, noone argued that proteins aren't 'weird little things' that often dont behave exactly according to general principles, and
that the course of a purification has to be adjusted individually. Again, though, general principles apply. Noone would start a purifcation by putting
it into a buffer at pH1, or 2 M ionic strength, or in the presence of denaturants or strong detergents at super high concentrations. Another general
recipe Exceptions apply, as usual, where some of the above conditions are
preferred.
Anyway... let's not start a fight here - you dont have to convince me on the ins and outs of protein purification, techniques and difficulties
thereof, I happen to purify different proteins on a monthly basis. I only once used A.S precipitation ever, but I would avoid it whereever I can. The
same goes for all precipitation techniques, in fact.
Peace
PS1 I believe all that Esplosivo meant to say was that if you have a buffer containing ammoniumcarbonate, or acetate, or sulphate, you will have
increasing acidity due to acid anion ((SO4)2-, (CO3)2-, CH3COO-), or the respective acid itself.
[Edited on 1-6-2004 by chemoleo]
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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PHILOU Zrealone
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NO NEED TO SHOUT!
-Yep...just standing it correctly for every body to avoid confusion and mixup...
Peace
-Always.
PS1 I believe all that Esplosivo meant to say was that if you have a buffer containing ammoniumcarbonate, or acetate, or sulphate, you will have
increasing acidity due to acid anion ((SO4)2-, (CO3)2-, CH3COO-), or the respective acid itself.
-I doubt it was that he meant with regards to what he wrote; but anyway he said something true; the only thing he got wrong is the conclusion...now
I'm sure he understands more the theory behind.If nobody tells you there is a problem; you could die making the same mistakes...to my feeling
it's better to tell someone he is wrong and why instead of let run and then allow misunderstanding or confusion to propagate.
I don't shoot at people...but yes sometimes my pedagogy looks harsh even if I tried to
bring some humour and vulgus pecus explanations
BTW: I have also spend many days in biochemistry labs (being a biochemical engineer explains this)...although I found biochemistry interesting maybe
because it is a small specific part of organic chemistry -I don't know?
I had quite some pleasure with enzymatic cinetic and gel permeation, dialyses, electrophoresis, sequençage, precipitation, isolations...but it
isn't as much fun and intensity as compared with organic synthesis and applications.
Where would biochemistry be without all those organochemistry tools and reactants???Maybe the funiest part for me is the understanding of the
reactions involved.
OK, to make phosphorescent plants glowing in the dark would have 100 % of my attention; so does a modified yeast that would help synthetising by 100%
specific reaction help....reduction of nitrocompounds to amino groups would be nice.
PH Z (PHILOU Zrealone)
"Physic is all what never works; Chemistry is all what stinks and explodes!"-"Life that deadly disease, sexually transmitted."(W.Allen)
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