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I accidentally left a delivery of NADPH Tetrasodium Salt (Santa Cruz Biotechnology) at room temperature for a little over half an hour.

In addition, when I opened up the package, I briefly held the bottle by the body instead of the cap (which would have prevented body heat from contacting it).

It was packed with airbags, but no ice packs, which is why I am asking this: is the powder still stable and usable?

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  • $\begingroup$ AliceD - I rolled-back part of your edit because I think the original meaning was lost. If you disagree, feel free to go back to your version. $\endgroup$ – canadianer Jul 30 '15 at 4:04
  • $\begingroup$ @canadianer - you're the expert on these matters :) I'll go for it :) $\endgroup$ – AliceD Jul 30 '15 at 4:05
  • $\begingroup$ @AliceD Well I wouldn't say that ;) Actually, whether the bottle was held by the body for a second instead of the cap hardly seems relevant to the question. $\endgroup$ – canadianer Jul 30 '15 at 4:07
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    $\begingroup$ @canadianer - yeah, I reckoned half an hour RT instead of -20C (dT=~45C) is a bigger deal than a second of bodyT-RT= 12C :) $\endgroup$ – AliceD Jul 30 '15 at 4:08
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    $\begingroup$ Since the delivery wasn't temperature controlled, the contents will have been at ambient temperature for essentially as long as it took to get from their warehouse to your site, which could be hours or even days. Is an extra half-hour likely to be significant? $\endgroup$ – David Richerby Jul 30 '15 at 8:41
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In my experience, half an hour at room temperature will make absolutely no difference. Boehringer Mannheim (now part of Roche), who at one time supplied the best NADPH, used to recommend storage at 4o C.

By A340 callibration, NAD(P)H is typically about 85% 'pure' based on dry weight measurements, and Sigma will try to tell you that the remainder is mainly water. That is, if you calculate a concentration based on the dry weight, the A340 will be about 85% what you expect.

Even if some water is absorbed from the atmosphere, this will make little difference as you are probably going to calculate the concentration based on the A340.

As User 243 has pointed out, if you are concerned that the NAD(P)H is degraded or oxidized, then an A340 measurement will probably set things right.

If you are concerned that all the A340 absorbing material is not NAD(P)H, or that a significant proportion is in the α-form (Oppenheimer & Kaplan, 1975) or is otherwise enzymically inactive, the best thing is an enzymic calibration.

This is done by adding limiting amounts of NAD(P)H to an assay system containing excess substrate and calibrating enzyme, and measuring the total decrease in absorbance (Racker, 1957).

A popular calibrating enzyme for NADH is aldehyde dehydrogenase (EC 1.2.1.3), as the reaction is practically irreversible, and it makes it easy to 'drive' the calibrating reaction to completing (and you don't have to worry about equilibria).

As I said in a previous post, NAD(P)H is UNSTABLE IN ACID (Oppenheimer and Kaplan, 1974). To put it crudely, 'the head (nicotinamide moiety) falls off', causing a bleaching of the absorbance at 340nm. Making 100mM acetate, pH 5, 100 micromolar in NAD(P)H produces a very significant decrease in A340 absorbance with time (which may be very easily monitored in a spectrophotometer).

Just because all the A340 material is due to NAD(P)H, this does not necessarily mean that the commercial preparation is pure. In fact, far from it. Dalziel (1963) showed that commercial preparations of NADH then available contained a competitive inhibitor of many dehydrogenases, most probably adenosine diphosphate ribose.

Accepting Dalziel's value of 17 600 M-1cm-1 for the extinction coefficient for NADH at 260nm, and assuming that NADPH has an identical value, then highly purified samples of NAD(P)H should have an A260/A340 ratio of about 2.83, and values higher than this may indicate the presence of impurities that absorb at 260nm, but not at 340nm. ADP-ribose is a prime example, but NAD(P)+ is another possibility.

I notice that in one of the links given by AliceD, Sigma quote a A260/A340 ratio of 2.32. So is the higher value due to the presence of residual NAD(P)+, or something else (a breakdown product?), or both?

You may need to use chromatography, typically on an anion-exchange resin, to obtain NAD(P)H free of impurities.

References

Dalziel, K. (1963). The purification of nicotinamide adenine dinucleotide and the kinetic effects of nucleotide impurities. J. Biol. Chem. 238, 1538 - 1543. [pdf]

Oppenheimer, N. J. & Kaplan, N. O. (1974). Structure of the primary acid rearrangement product of reduced nicotinamide adenine dinucleotide (NADH). Biochemistry 13, 4675 - 4685.[pubmed]

Oppenheimer, N. J. & Kaplan, N. O. (1975). The alpha, beta-epimerization of reduced nicotinamide adenine dinucleotide. Arch. Biochem. Biophys.166, 526 - 535.

Racker, E. (1949). Aldehyde dehydrogenase, a diphosphopyridine nucleotide-linked enzyme. J. Biol. Chem. 177, 883 - 892. [pdf]

Racker, E. (1957). Spectrophotometric enzymatic methods for the determination of aldehydes and ketoaldehydes. Methods Enzymol. III, 293 - 296.

Other References

Ciotti, M. M. & Kaplan, N. O. (1957). Procedures for determination of pyridine nucleotides. Methods Enzymol. III, 890 - 899

Horecker, B. L. & Kornberg, A. (1948). The extinction coefficients of the reduced band of pyridine nucleotides. J. Biol. Chem. 175, 385 - 390. [The definitive determination of the extinction coefficient at 340nm] [pdf]

Kornberg, A. & Pricer, W. E. (1950). On the structure of triphosphopyridine nucleotide. J. Biol. Chem. 186, 557 - 567. [This paper shows that the 'extra' phosphate in NADP(H) is attached to the 2' position of a ribose. In Coenzyme A, of course, the phosphate attached to the ribose is on the 3' position. [pdf]

Pullman, M. E., San Pietro, A. & Colowick, S. P. (1954). On the structure of reduced diphosphopyridine nucleotide. J. Biol. Chem. 206, 129 - 141 [pdf]

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You could measure OD at 340nm. If OD340 is much lower than expected, NADPH is oxidized and does not have much biochemical activity.

http://www.bmglabtech.com/media/35216/1043734.pdf

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  • $\begingroup$ In addition to oxidation, it is probably worth mentioning that the acid rearrangement product does not absorb at 340nm. $\endgroup$ – user1136 Jul 31 '15 at 8:36
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I think when they sent it to you without dry ice, it is probably OK to store it at room temperature.

Sigma seems to advise on their NADPH to store dehydrated NADPH at room temperature, while they advise to store the hydrated forms at -20oC. Likely the presence or absence of molecular water in the material is crucial for its storage.

disclaimer: I am not an organic chemist and when you are doing crucial experiments with your material and you doubt its quality, I'd recommend to chuck this batch away and order new. Likely, a single day of futile experimentation in the lab due to bad reagents is more expensive than the purchase of another jar of NADPH.

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  • $\begingroup$ What exactly is the difference between 'hydrated' and 'dehydrated' NADPH, other than water or hydration, and how does this give rise to differences in stability? I see no water molecules in either of the Sigma diagrams you link to. $\endgroup$ – user1136 Jul 31 '15 at 8:43
  • $\begingroup$ @TomD - the water in NADPH-hydrate is not considered part of the molecule. It is part of the crystal. Therefore it doesn't show up in the formulas. The stability difference is because NADPH reacts with water I guess. The dehydrated form is called the anhydrate. I just talked to a chemist about this :) And I linked the question to him too. btw: I +1'd your answer. Great stuff. $\endgroup$ – AliceD Jul 31 '15 at 10:17
  • $\begingroup$ The question I posed is what is the difference between hydrated and unhydrated NADPH other than water of hydration. I am aware of the distinction that your chemist friend makes. You say NADPH is unstable in water. What is the breakdown product? Does it cause bleaching of the A340? The only reaction of NADH with water that I know of is the enzymic hydration of the 5,6-double bond. $\endgroup$ – user1136 Jul 31 '15 at 10:41
  • $\begingroup$ @TomD - quite frankly I don't know, and my friend did neither. The only clue I got was the fact that the hydrate salts need storage at -20oC, the anhydrate form not. I'm not a chemist (as posed in the disclaimer :-) $\endgroup$ – AliceD Jul 31 '15 at 10:44

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