Lidstrom:Solution Stock Info

Back home: Lidstrom:Protocols

ATP

• Mila said ATP doesn't degrade into ADP or AMP, and ADP doesn't degrade into AMP either. Enzymes are required for these interconversions. You can keep stocks at -20oC. -JM 12/17/2013

Recommendations from 1993 book below

• ATP, sodium salt (MW 551)
  • Store desiccated powder @ -20°C
  • In H2O, bring to pH 7, with 2 equiv NaOH, store -20°C
  • Stable for months with minimal concentration loss

NAD(P)/H

• When NADH degrades, it turns yellow. It is not degrading into NAD, though; it degrades into something else. From Mary & Mila 12/17/2013 -JM

• NAD and NADP are stable if stored dry and in the dark. Solutions of NAD(P)+ are colorless and stable for about one week at 4 °C with neutral pH, but decompose rapidly in acids. Upon decomposition, they form products that are enzyme inhibitors. (source)
• Under acidic conditions, stability is lowered significantly. (reference)
  • "2.1.4.1. Reduced coenzymes (β-NADH and β-NADPH)
    • The reduced coenzymes are affected by two major degradation mechanisms: (i) oxidation to β-NAD+ and β-NADP+ by dissolved molecular oxygen and (ii) an acid-catalyzed, first-order, three-step consecutive mechanism involving the addition of water, anomerization and cyclization [60], [61] and [62] (Fig. 1). The latter mechanism is the main contributor to β-NADH/β-NADPH degradation. Although ideal storage conditions for β-NADH and β-NADPH have been described in some detail [62], [63] and [64] the overall stability is very poor in aqueous solutions. The highest stability of β-NADH was reported for a 50 mM Tris-EDTA-HCl-Buffer, pH 7.7, in which the half-life at room temperature was 4 d. Under acidic conditions, stability is lowered significantly. At pH 4 and T = 41 °C, for instance, β-NADH has a half-life of only 5.5 min [62]. Solutions containing β-NADH should thus always be adjusted to a pH around 8 [62]. Notably, several common salts such as sodium oxalate, sodium sulfate, disodium hydrogen phosphate and sodium maleate were shown to catalyze β-NADH degradation [62], [65] and [66] and should hence also be avoided.
    • The phosphorylated β-NADH analogue β-NADPH is generally even less stable than β-NADH [62]. At pH 6 and T = 41 °C, for example, β-NADH had a half-life of 400 min while the half-life of β-NADPH was only 56 min [62].
    • A significant improvement in stability can be achieved if β-NADH is stored in water-free solvents as this avoids degradation by the acid-catalyzed mechanism. β-NADH was stable for several months if stored in Tris-buffered ethylene glycol at 4 °C [64].
    • It may be noted that the acid-catalyzed degradation mechanism yields acid-stable end products for β-NADH and β-NADPH, respectively (Fig. 1). As these end-products are not yielded from the oxidized forms (β-NAD+, β-NADP+), they could, in principle, be used to indirectly determine the level of β-NADH and β-NADPH present upon sampling [61] and [67]. However, we are currently not aware of quantitatively validated methods making use of this approach."

From 1993 book referenced below

• NAD(P)H are rapidly destroyed in acid in conditions where NAD(P)+ are completely intact
• 99% destruction of NADH
  • @23°C = 1.2 min @ pH 2, = 2 hr @ pH 4
  • @38°C, 3-4x faster
  • @60°C, 20x faster
• NADPH is destroyed faster than NADH (~80% faster @ 30°C)

• @pH 2, NAD+ is 100,00x more stable than NADH
  • Allows elimination of reduced form by pH drop
• Above pH 7, degradation rate of NAD increases,5000x faster at pH 12.5-13
• NADP+ appears to be more stable than NAD+
• Higher temp, more salts increase degradation rate

• NADH becomes more stable with pH increases, excluding low concentration in small volumes
• Oxidation is a problem above pH 8
• @ pH 7 25°C, rate of destruction in ~0.2%/hr, increases 10x @ pH 6

• Strong solutions (40 mM) of NADH store well @ pH 9-12, but large losses @ 4°C
• Weak solutions (0.4 mM) of NADH store well @ 4°C from pH 9-11, destroyed @ -20°C pH >10.5
• Similar characteristics for NADPH

• NADH presence in small volumes is sensitive to oxidation at neutral pH
  • increases ~inverse square root of the volume
  • can prevent with 1 to 2 mM ascorbic acid

Recommendations

• Store pyridine nucleotides, desiccated @ -20°C
• Can store NAD+/NADP+ solution for months without significant concentration change/
• Can standardize stock solutions with spectrophotometer
• Dissolve NAD+/NADP+ in water, ~100 mM
• Unless < -40°C, NAD(P)H should be prepared at < 5 mM, pH 9-11 and stored at 4°C. At low temp, strong solution can probably be stored without too much loss

CoA

• From The Stability of Coenzyme A, 1954:
  • Acetone powders and high purity CoA preparations in the free acid form were found to be stable for several years when stored under dry conditions, at room temperature.
  • Drying of high purity powders in vacuo produced marked decreases in activity.
    • The effect of drying high purity powders in vacuo over P₂O₅ is shown in Table 11. Destruction increases rapidly with temperature, but there is little additional loss after four hours.
  • Aqueous solutions subjected to autoclave temperatures showed considerable destruction.
  • Alkaline solutions of CoA were found to be unstable, while acidic solutions were much more stable and even showed an increase in activity under certain conditions of time, temperature and pH.

• From Sigma
  • Acetyl CoA is soluble in deionized water at 100 mg/mL. Acetyl CoA is stable in neutral and moderately acidic solutions but will hydrolyze in alkaline and strongly acidic solutions. Aqueous solutions stored in single-use aliquots are stable for up to two weeks at −20°C and up to 6 months at −80°C.

• From Avanti
  • A stock solution may be prepared by dissolving the CoA in distilled/deionized water or buffer that has been sparged with nitrogen to remove oxygen (heat and/or sonication may be necessary to dissolve CoA). CoA’s are soluble in water to < 50mg/ mL. The aqueous solution should be stored at 2-8°C and used within 1 day. CoA’s are not stable in aqueous solution and will degrade rapidly when stored in water. For long term storage, Avanti recommends that CoA’s be stored as a powder at -20°C. The product should be stable in this form for at least 1 year.

CoA Conclusions

• Powder is pretty stable
• Solutions probably shouldn't be subjected to freeze/thaw
• Don't put them in alkaline solutions.

Citations

Passonneau, J. V., Lowry, O. H., & Lowry, O. H. (1993). Enzymatic analysis: A practical guide. Totowa, NJ: Humana Press.