I have a solid urease, how can I prepare a suspension of this enzyme containing 3.5 kU/L?
You have to know the activity of solid urease (units/mg of protein) and the purity of the urease. See the label or urease specifications
Estimado Profesor Francisco Solano, muchas gracias por su disposición de ayudarme con esta interrogante. En el datasheet del producto solo me dice que tiene un contenido de 0.9-1.4 mg de proteína/vial. No conozco la actividad. Que debo hacer ahora?
Hola Livan, gracias, para mi es un placer. Pero necesitamos saber la actividad especifica, y cuantas unidades tienes por mg de proteina, Sin ese dato, es imposible preparar una disolucion en Kunidades por Litro. De donde procede la ureasa? s comercial? Dice algo de katales en el datasheet? Busca algo mas..... seguimos en contacto
Por tener alguna idea o referencia, buscando "urease specific activity", encuentro lo que pego abajo. la ureasa de A. niger cuando se purifica (4126 veces) puede llegar a tener 1341 unidades/mg proteina. Pero ten en cuenta que si no se purifica tanto, la actividad especifica sera menor. Espero que eso te pueda ayudar
Journal of General Microbiology (1993), 139, 957-962. Isolation and characterization of urease from Aspergillus niger PATRICK T. SMITH,* A. DOUGLAS KING JR and NELSON GOODMAN
Urease was purified (4126-fold) from Aspergillus niger (NRRL 003) to a homologous enzyme preparation with a specific activity of 1341 micromol min-' (mg protein)? O
Thank you Vijayaraghavan, we agree. The problem is that the label does not supply that information (specific actiivty). Have you used that jack bean urease?
These are good news!. So, you mean that the specific activity should be in the range from 100-200 units/mg. Provided that range, it is easy to get a solution around 100-200 unit/ml by dissolving the solid in 1 ml of appropriate buffer and then, the desired concentration by dilution. I am sure that Dr. Livan Delgado will see this. Anyway, thanks a lot.
In addition to the units/mg, you'll need to dissolve your powder in water and add ammonium sulfate to create the desired 3.5kU/L in a stable suspension you can store at 4C.
Dear colleague, I so grateful for your certain and useful commentaries, I will prepare the urease solution as advised Dr. Vijayaraghavan, also I would like to acknowledge to Dr Solano and Dr Eric van der Graaff. Best regards to all.
Thank you Eric. To finish, I suppose that ammonium sulfate is to get enzymatic stabilily, but could you write the needed concentration? As you know, ammonium sulfate is very soluble. Thus Dr. Livan will have all data.
Dear Dr Erick, could you send me please the concentration of ammonium sulfate to stabilize the urease solution...thank you again.
The ammonium sulfate ensures stability of the enzyme upon storage at 4C. In general, repeated freezing and thawing of enzyme in water severely reduces enzyme activity. The final concentration of the ammonium sulfate should be 3.2 M ammonium sulfate at pH 6.0. In practice, we could not come above 2.9 M, so dissolved the (lyophilised) powder in very small volume of water and added this 2.9M stock. The enzyme will precipitate, forming a suspension that appears milky, so you should mix well before pipetting. When diluting this for your assay mix, the protein will dissolve properly.
Good luck!
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