[cityqis (OP)]

Hi everybody..

Well im more interested in the enzyme area as im doin a research on a plant enzyme..The problem is this, as im testing the pH stability, it turns out that after a period of time my residual activity turns out to be higher than the initial activity which makes the residual activity more than 100%..
Is this possible..? Isnt the activity suppose to decrease after a period of time..?
CAn anybody shed any light on this matter....? pleaase...

[MayoFlyFarmer]

can you explain a little more clearly/in depth exactly the experiment you are trying to perform?

[cityqis (OP)]

Hi...
Thanks 4 ur reply. Here's some info on the experiment. i'm trying to figure the pH stability of this new plant protease and the range of pH starts from 4 up to 8. Basically, i just incubate the enzyme in these different buffers at 4•C and check the initial activity and the activity after 6h, 24h, 48h and 72h... I'm expecting the activities to decrease after a period of time. However, it turns out that the enzyme activity in all buffer (pH 4 to 8)seems to be higher than the initial reading.This is rather suprising as I never read any journals or papers which had recorded such findings []..Is the info sufficient?  [] Just tell me if u need more info on my research..

[cityqis (OP)]

Hi...
Thanks 4 ur reply. Here's some info on the experiment. i'm trying to figure the pH stability of this new plant protease and the range of pH starts from 4 up to 8. Basically, i just incubate the enzyme in these different buffers at 4•C and check the initial activity and the activity after 6h, 24h, 48h and 72h... I'm expecting the activities to decrease after a period of time. However, it turns out that the enzyme activity in all buffer (pH 4 to 8)seems to be higher than the initial reading.This is rather suprising as I never read any journals or papers which had recorded such findings []..Is the info sufficient?  [] Just tell me if u need more info on my research..

[MayoFlyFarmer]

what sort of test are you sing to check for enzymatic activity?

[cityqis (OP)]

the method used is as described by Wang et al (1974)..It involves incubating the enzyme with the substrate (i used 1% Bovine Serum Albumin)and incubating it at 38°C for 20 minutes. The products which is the L-tyrosine is detected through the UV-VIS spectrophotometer at 280 nm...

[MayoFlyFarmer]

hmmm...  that is puzzling.  Could it be that during the incubation period the reaction acclimates to 4-degrees at which the enzyme may be more efficient?  or that the uv bulb in the spec has chance to warm up more during that time thus throwing off your readings?

do you know the pH of the cellular environment from which this plan comes from?  could it be that the activity of the enzyme is supressed by pH in its native environment, and when you put it into different pH buffers, it takes time for it to aclimate to that pH??

i dunno, these are all kind of long shots, but as you pointed out, its not what you would expect to see.  if you figure it out post back.  i'd be curious to see what the issue is.

[cityqis (OP)]

yeah..its puzzling (sigh).. one thing that came across my mind was, could it be there's contamination from microorganism hence somehow contributed to the protease activity []..but then again..6 hours of incubation might be too short a time to allow any growth of microorganism...well, its ok... i'll look further into this matter..who knows..it'll be a great discovery.. [] (keeping myself optimistic)

[cityqis (OP)]

Hi again..!!
Guess what..i think i know what the problem is.. I found an article saying that the polyphenols in plant do contribute to the reading of absorbance at 280nm.. []..My enzyme gets darker as the incubation period lengthen..of course they are contributed by these polyphenols..!! haha..why didnt i think of that...  []Therefore im expecting to see a different result after enzyme is purified ..anyway..thanks for your ideas..i really appreciate it..

[MayoFlyFarmer]

that makes sense.  i'm curious to hear how it works out for you after purifying....