Hi

Please see this paper

http://jcb.rupress.org/content/17/1/19.full.pdf

A few suggestions: (1) Increasing the viscosity with high concentrations of sugars will slow down the reaction by reducing diffusion rates. (2) Minor contaminants in the mannitol, such as metal ions, could be inhibitory, rather than mannitol itself. (3) Mannitol may be acting as a weak competitive inhibitor if it resembles any of the substrates. (4) Mannitol may be interfering with product detection, rather than enzyme activity. (5) Mannitol may be binding up one or more of the substrates, reducing the effective concentration of the substrate(s). (6) If you want to try other sugars as stabilizers, I recommend trehalose at concentrations up to 0.5M.

Which enzyme are you working with? Are you working with any enzyme that uses mannitol as substrate? Mannitol is often used for inducing water/drought stress in plants. Mannitol has potential to retain water with itself and also may retract water from living systems. It is likely that at higher concentrations mannitol might be interfering with 3-D structure of your enzyme by influencing its water hydration capacity. To the best of my knowledge, for optimal enzyme activity it is important to precisely regulate water potential. I am sure, as expert in physical chemistry of proteins can address to your question more authentically.

Gisele:

Are you using a mannitol containing buffer?

Gisele:

See page #3 in the following article (New Phytologist).

http://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1986.tb00583.x/pdf

New Phytologist

Volume 102, Issue 2, pages 285–291, February 1986. Article first published online: 2 MAY 2006.

DOI: 10.1111/j.1469-8137.1986.tb00583.x 

Gisele: I am assuming that you are using mannitol solution to preserve your enzyme. Mannitol scavenges hydroxyl radicals. Perhaps at this concentration, mannitol scavenges hydroxyl radicals in your solution.

Thank you for all the answers.

My enzyme is an L-amino acid oxidase. These enzymes are very labile (I don't know why). So I can't freezinf and freeze-drying. At 4ºC it keeps a little its enzymatic activity but decreases in the course of time. 

I am evaluating its activity during 40 days (10 days until now) and in the presence of different concentrations (1.4%, 2.8%, 5.6% and 8.5%) of posible adjuvantes (mannitol, sucrose, L-Lys and L-Gly). At time zero, all activities were the same as the control (without "stabilizer"). In the course of time, L-Lys and L-Gly were not enough ti keep the enzyme activity. Until the 10th day, only 8.5% is able to keep the enzymatic activity. On the other hand, only 2.8% of mannitol preserve L-amino acid oxidase active and in 5.6% and 8.5% of this same suggar the activity decreases (as I said in the question)

L-amino acid oxidases are flavoproteins. Perhaps your enzyme is losing its flavin cofactor. You might be able to stabilize it better by adding some FAD or FMN, whichever flavin is normally present.

Since flavins absorb visible light, you should minimize exposure of the protein to light.

Another way you could try storing it is in 50% glycerol at -20C. The glycerol keeps the enzyme from freezing.

With properly chosen co-solvent you can "regulate" a little the enzyme reaction rate (see e.g. 61. Bérczi, A., Møller, I.M. (1993) Control of the activity of plant plasma membrane MgATPase by the viscosity of the aqueous phase. – Physiol. Plant. 89: 409-415.). Flavo-proteins in general need light protection for keeping them "alive".

I've already been keeing the enzyme with ligh protection. 

I saw 2 papers that say that it doesn't loss FAD or FMN. The authors discuss that is a shift in the spectrum absorbance, but FAD keep together LAAO.

Thank you, Adam and Alajos

There appear to be mechanisms for the stabilization of the flavin prosthetic group of LAAO. The adenosyl group of the FAD containing L-AAO is found in a certain orientation in both prokaryotic and eukaryotic LAAOs , and is stabilized by a number of hydrogen bond interactions. 

At a concentration of 2.8% of mannitol the activity is kept. It might imply  that the FAD prosthetic group  was not impaired at this concentration.

Thank you, Chithan. Do you know why this decreasing happens? I mean, the first thing that I thought is that increasing the amount of mannitol, the activity would also increasing or keeping at the same level not decreasing. I think its not a problem in mannitol solution since in time zero the activity is the same as the control (without manitol). In course of time this decreasing happens

Maybe mannitol ia able to do hydrogen bonds with enzyme and impaired the bonds between FAD and LAO?

Hi Gisele,

One way to store sensitive enzymes is to aliquot them into 1.5 mL cryotubes and then snap freeze them in liquid nitrogen and store at -80. Then only thaw the frozen tubes one time.

As for the maninitol concentration, mannitol does bind water tightly, as you increase the concentration it may reduce the available water for the enzyme such that it cannot catalyze the reaction.

Hi Gary,

Thank you so much for the information. Do you have some paper or just the reference that explain the effect of mannitol you said?

Hi Gisele,

Wikipedia, which described its use in osmotherapy and the structure which indicates it can make numerous (12) H-bonds which would create a shell of water around it.

https://en.wikipedia.org/wiki/Mannitol

Gisele:

Good point!

As you have stated, L-lysine and glycine are not enough to retain enzyme activity. You need osmolytes (mannitol and sucrose) to preserve enzyme activity. Perhaps at a mannitol concentration (double) of 5.6%, your oxidase is becoming denatured (mannitol/enzyme ratio is higher). The question of whether there is any protease contamination in your enzyme preparation arises. The carbohydrate moieties of the dimeric glycoprotein, LAAO, are considered to increase the solubility and viscosity of the protein and maintain the stability of electrical charges; the glycans are also considered to protect the enzyme against potential proteolysis.

Mannitol's effectiveness in preserving the native structure of proteins in known. Mannitol is also known to rapidly crystallize out of solution. Mannitol, in its amorphous form, has been used for protein stabilization in presence glycine. . The crystalline form of mannitol may be decreasing enzyme  stabilization (causing denaturation of the enzyme). Sugars/sugar alcohols affect secondary structure of proteins. Sugars protect proteins during dehydration by hydrogen bonding to the dried protein, thus serving as water substitutes. In your case, we do not know if there is a ratio of the enzyme to mannitol that facilitates optimal stabilization of L-AAO. 

Gisele:

Enzymes can be stabilized by hydrogen bonding. At a concentration of 5.8% mannitol, the secondary structure of your protein preparation, L-AAO, could have been impaired. Hydrogen bonds in the folding of proteins have been connected with osmolyte-induced protein stabilization. Sugar osmolytes, depending on concentration, promote folding. Osmolytes are thought to stabilize proteins by modifying hydrogen bonds in the protein hydration layer. If L-cysteine residues are modified in your enzyme that would cause protein unfolding. Dithiothreitol and EGTA addition to your system may aid stabilization. 

Gisele:

Some links of papers, which could be of interest:

Arch Biochem Biophys. 1999 May 15;365(2):289-98. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Allison SD(1), Chang B, Randolph TW, Carpenter JF.

http://www.ncbi.nlm.nih.gov/pubmed/10328824

Carpenter, J. F., Crowe, J. H., and Arakawa, T., 1990. Comparison of solute-induced protein stabilization in aqueous solution and in frozen and dried state. J. Diary Sci. 73, 3627-3636.

http://www.journalofdairyscience.org/article/S0022-0302%2890%2979065-0/pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1302615/ glycerol

Carpenter, J. F., Prestrelski, S. J., and Arakawa, T. 1993. Separation of freezing-and drying-Induced denaturation of lyophilized proteins using stress-specific stabilization. Achives of Biochemistry and Biophysics. 303, 456-464.

books.google.co.in/books?isbn=0306467410

http://www.ncbi.nlm.nih.gov/pubmed/8512328

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784861/

http://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1986.tb00583.x/pdf

Ru'WART M J & SuELTER C H (1971) The

Journal of Biological Chemistry, 246, 5990-5993

Cleland, J. L. and Jones, A. J. S. (1996). "Stable formulations of recombinant human growth hormone and interferon-gamma for microencapsulation in biodegradable microspheres." Pharm. Res. 13: 1464-1475.

http://www.ncbi.nlm.nih.gov/pubmed/8899836

Thank you so much, Chithan. 

I am reading them!

Hi

Please see this paper

http://jcb.rupress.org/content/17/1/19.full.pdf