Alpha-ketoacids absorbance

[magrathea]

Hi everyone.
I know that pyruvate has an absorbance maximum at 320nm.
I would like to know from a chemist if this is a general property
of alpha-ketoacids. Anyone has a refcerence to that? Or better
a link to a "free" absorbance spectra database?
Thanks in advance,
Gianluca

[tgd]

Hello,

Well,I must say this is news to me!

Are you sure pyruvate absorbs with a maximum
at 320nm?

This seems very high and is perilously close to 340nm, the routine wavelength for NADH-linked enzyme assays. If pyruvate but not lactate absorbs at 340 nm, would we not have to apply a correction factor to the extinction coefficient of 6220 M-1.cm-1 when assaying lactate dehdrogenase?

The usual assumption is that the only contributor to the delta-A340 is the NAD+/NADH half reaction. This is not true in all cases, of course: dihdrofolate reductase is an obvious exception. However, I have never seen a correction applied to the lactate dehydrogenase assay.

It is, of course, a very interesting question. Perhaps you could post your source of information?

Good luck,

TGD.

An afterthought: Are you referring to the enol form of pyruvate? As far as I remember, the enol form in solution at neutral pH is a lot less than 1%. However, you may be referring to a pH very different from neutrality!

[magrathea]

Originally Posted by tgd

Hello,
This seems very high and is perilously close to 340nm, the routine wavelength for NADH-linked enzyme assays. If pyruvate but not lactate absorbs at 340 nm, would we not have to apply a correction factor to the extinction coefficient of 6220 M-1.cm-1 when assaying lactate dehdrogenase?

Well, I suppose it's just a matter of numbers: the extinction coefficient
of pyruvate @320nm is much lower in respect to NADH: 21 M-1.cm-1 so,
in an equimolar reaction with NADH, it will account only for less then 0.3% of
absorbance variation near 320-340 nm.

Originally Posted by tgd

It is, of course, a very interesting question. Perhaps you could post your source of information?

I determined the pyruvate extinction coefficient by a titration @ pH 7.0.
(I can send you some spectra if you are interested in).

Cheers,
gianluca

[tgd]

Hello,

You are, of course, perfectly right, and thank you very much for that interesting piece of information. When you spectra are published, I would like to get the reference. Do I take it that 100mM pyruvate at neutral pH has an absorbance of about 0.2 at 320 nm? (Do you know the extinction coefficient at 320nm?)

Thanks,

Tgd

[magrathea]

As reported above, the ext. coef. @ 320 is 21 M-1cm-1, e.g.a
0.1 M pyruvate solution adsorbs about 2.1 Abs. Units.
I attached the spectra of the titration (I'm sorry,
but the axes labels are not in english).

All the best,
GM

[tgd]

Thanks!

Those spectra are very nice, and I have downloaded your pyruvate.jpg. I hope this is OK

TGD

PS I suppose you know about Pocker's work on pyruvate? (it is a while since I have read these).


Pocker, Y. & Meany, J. E. (1970). The reversible hydration of pyruvic acid. II. Metal ion and enzymatic catalysis. J. Phys. Chem. 74, 1486 - 1492

Pocker, Y., Meany, J. E., Nist, B. J. & Zadorojny, C. (1969). The reversible hydration of pyruvic acid. I. Equilibrium studies. J. Phys. Chem. 73, 2879 - 2882

I have pdf's of these if you are interested (but cannot post to a bulletin board due to copyright). Send me an e-mail, maybe.

[thenewday007]

I am a newer here.Glad to meet u.Is anyone online?

[aion12]

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