Do enzymes really need to be "frozen"?

[Phil_]

I've been puzzled over how a small lab could store their enzymes. Even in the US, the power goes out for at least a day about once a year. As we "all know", enzymes must be stored at -20F, kept in an ice bucket while you're using them, and returned as soon as possible to the freezer. It would be a major blow to a small lab if all their enzymes were destroyed once a year by a power outage.

This paper claims that all the restriction enzymes they tested maintained their activity at room temperature for a week, and most maintained activity for a full year:
http://www.biotechniques.com/multimedia/archive/00011/00293st06_11772a.pdf
(The file may be attached if that google groups feature really works.)

What do you think? Is it okay to just let your enzymes warm up when the power goes out?

(A related question is: How low a storage temperature can you use on enzymes in a 1:1 glycerol suspension without freezing them?  -20 is okay; how about -30?)

[Avery louie]

They document is attached-thats neat!

Fortunately, you freezer does not heat up too much if it is well insulated and has a lot of thermal mass.  And unfortunately, the enzymes in the article look like they were dehydrated and dessicated- most of the enzymes I purchase are already in solution.

Still, it is something to think about for shipping- which can be as expensive as the enzyme itself, if you are not careful.

--A

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[Avery louie]

I take it back- I think they might have been in solution.  interesting!

[Dakota Hamill]

Wow that's a great looking report.  I just glanced over the gel pictures and it was cool to see the enzymes still working well after a year!

I'd just venture a guess that the low temperature would help slow degradation of the enzymes IF they happened to be contaminated with protease.  But anyone working with any level of care could avoid that contamination, and hence...maybe their sample would still have functional enzymes if they forgot it on the lab bench at room temperature for a while.

Pretty interesting though.

Has anyone done a similar report but with polymerases?

[Avery louie]

The Taq I ordered from openbiotech did 2 days at rt and was fine- no Suprise since it is stable at high temperatures, and hopefully was protease free.

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[Mega]

Have to revive this old discussion... 

"This paper claims that all the restriction enzymes they tested maintained their activity at room temperature for a week, and most maintained activity for a full year"

I always thought (and heard from a lab worker) that restriction enzymes need ATP  and therefore the entire solution has to be kept at -20°C to prevent ATP decay...

But didn't read that they added ATP then? Is the ATP story wrong? 

[Cathal Garvey]

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I'm not sure, but I don't think most restriction enzymes need ATP,
though many other types of enzyme do.

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[Philip Goetz]

Can anyone list some enzymes that need to be at -20?
Ones that we need to worry about if the power goes out for a couple of days.

[Mega]

Really? But what powers them then? just the energy from room temperature? 

So, basicallly EcoRI and HindIII are very stable it seems. 

1. For EcoRI, no

appreciable decrease in activity was

noted until eight weeks after transfer to

37°C, and some activity still remained

after 12 weeks (the absence of activity

of the 4°C sample after two weeks is

most likely due to experimental error

because subsequent reactions with the

same enzyme exhibited activity).

HindIII appeared to be even more stable, and it was not until six months after transfer to 37°C that a reduction in

activity could be seen. For SmaI, although no activity was noted after storage for one week at 37°C, this is again

likely to be due to experimental error

because a similar result was observed

with the -20°C sample for digestion at

this time point. Reduced activity was

subsequently noted after two and four

weeks of incubation at 37°C, although

none remained after eight weeks.

[Andreas Sturm]

There are three different types of restriction enzymes. Type I cuts DNA at random locations as far as 1000 or more base-pairs from the recognition site. Type III cuts at approximately 25 base-pairs from the site. Types I and III require ATP and may be large enzymes with multiple subunits. Type II enzymes, which are predominantly used in biotechnology, cut DNA within the recognized sequence without the need for ATP, and are smaller and simpler. 

http://biotech.about.com/od/proteinengineering/a/restrictenz.htm

So no ATP for molecular cloning restriction enzymes!! 

[Josiah Zayner]

Storing enzymes in glycerol in a freezer is all about thermodynamics. When a protein(enzyme) are at higher temperatures it has access to a wider variety of conformational states it can inhabit according to Boltzmann and physics. What this means is that the protein can cross over energy barriers into disordered or non-functional conformations. This is why proteins are always stored at or around -20C in glycerol. You don't want to freeze proteins because the changes in the organization of water can effect the structure and conformation of the protein. You do not want to store proteins at -80C because proteins can also cold denature http://waterinbiology.uchicago.edu/Papers_files/fluc-cold_jp075928t-PJR.pdf . So the complete coldest temperature might not be the best. It is very reasonable to think that some proteins can last a long time at room temperature but in general it is better to store at 4C or at -20C in glycerol.

-20C is your average freezer and 4C is your average refrigerator. If one cannot store at -20C then store at the next lowest temperature 4C, this will help increase the lifetime of the protein. 

A single Power outages should not drastically effect the lifetime of your enzyme. Taking the enzyme from -20C to ~room temperature often would probably effect it more if one is not using it on ice or even if one is using ice.

Storage guidelines for things in molecular biology are general guidelines for safe and practical science. Obviously these rules can be broken just understand why they can be broken and when it is reasonable to break them and everything will be ok. However, as a general guideline for beginners one should be as uptight as possible becuase it makes troubleshooting that much easier.

[Nathan McCorkle]

On Sun, Mar 24, 2013 at 6:54 AM, Mega <masters...@gmail.com> wrote:
> Really? But what powers them then? just the energy from room temperature?

enzymes are catalysts, meaning they simply lower the activation
energy. Whether the remaining energy is thermal or chemical (ATP)
comes after the fact. You could twist this into perspective by
thinking their 'power' input was during their synthesis, the energy
consumed to build the amino acid chain, and in all the reactions that
lead to that sequence of amino acids being generated. In actuality the
way they work is a beautiful sequence of magnetic-like push/pull
actions on electrons of the target molecule by specific atoms on the
enzyme. The push/pull of electrons is sequenced that some area of the
molecule becomes reactive (or less un-reactive). In simple cases that
reactive area is where you want the work to be done (adding a
molecule, cleaving one, pulling electrons off or pushing them on), in
more complex cases this area simply influences yet another area of the
target molecule in some way, which is where you want the work done.
Depending on the enzyme and target molecule and target operation this
sequence can continue many more times, and the sequence of
interactions would take place in both the target molecule, the enzyme,
cofactors, the local solvent shell and even the electromagnetic
potential across the wider reaction zone cross-section.

--
-Nathan