[tezla93]

Hi guys first post here, hopefully of many!

Pooled data from a FluroMetPlate assay testing the inhibitory effects of Mercury, Zinc, Cadmium and Copper on Beta-galactosidase has shown up (what I think to be) some strange results. Although all of the metals are toxic to the enzyme at high enough concentrations, zinc and cadmium actually increased enzyme activity at very low concentrations.

My literature search has proven fruitless but I could just be looking in the wrong places. Is the data to blame? or is there a painfully obvious answer that is just eluding me? any research for me to reference would also be much appreciated, or a point in the right direction!

[tezla93]

*Ignore me, I am impatient*!

[Babcock_Hall]

It is a forum rule that you must show your attempt to solve a problem before we can help you.  What literature searches did you do?  Are you working with the E. coli enzyme?  What are some of the essential residues of beta-galactosidase?

[tezla93]

Its well known that heavy metals can disrupt the tertiary structure of enzymes that contain sulfhydryl bonds that are critical to the structures integrity. Without these the enzyme can't do its job! What I cant get my head round is exactly why they are actually increasing the enymes activity at low concentrations even though they should be disrupting its structure

Edit: Yes I am working with the E.coli enzyme. Our assay was performed in vitro using a a purified enzyme.

Edit: I've been having a think about it for days now but still haven't gotten much further. I've though that the assay could provide an explanation as most toxicity research I have found tests the enzymes activity inside the living system. Or perhaps it could be due to E.coli actually needing small amounts of zinc to be"healthy", making it logical to assume that its enzymes are structurally suited to being more active in similar environmental conditions. Either way I can't find any papers to confirm my suspicions so I assume I'm just wrong!

[Babcock_Hall]

What do you know about the metal ion dependence of this enzyme from the literature, and what searches have you done?

[tezla93]

Never came across the term "metal ion dependence" in my first year studies! =0 However I have read that beta-galctosidase requires Mg as a co-factor and to a lesser extent Na.

Most of my literature searches have been around similar experiments as mine; testing the effects of heavy metals on beta-galctosidase activity from E.coli, other bacterium or just from the purified enzyme. They have all agreed on the toxicity of the four metals and are concurrent with my findings of mercury being the most toxic. However none have shown any evidence that at low concentrations that zinc and cadmium can actually cause the enzymes activity to increase.

[Babcock_Hall]

My working hypothesis might be that some metal ions can substitute for Mg (II) at low concentrations.  There is also a cysteine residue that could possibly be affected by the presence of a metal ion.

[Arkcon]

However none have shown any evidence that at low concentrations that zinc and cadmium can actually cause the enzymes activity to increase.

Is the increase significant?

Note:  I don't want just a yes/no answer.  I want you to define what is a significant result, how you've determined it is significant, and I want to know that all of your data:  the blank, the positive control, the negative control, the standards, and your toxic samples are significant.  A table of numbers would probably be the best.

[tezla93]

If you insist. Already made the table last week. Just take a look for yourself and you can easily see that the %inhibition in the negative figures is significant

Note: I Pm'd you the table

[Arkcon]

Why not post the table for everyone to help.  I'm at work, and I don't really have time to review it.  Also, it is your responsibility -- I only mentioned I because I suspected that you didn't have it at all.  Now, you can compare for all of us ... Your blanks, controls. Positively be and negative results.

[tezla93]

The table shows up messy in here but basically I've recorded a near 20% further activation of beta-galactosidase in relation to my control by zinc at a concentration of 0.0033ppm.

But why would you think I didn't even have any data when my opening post talks about the pooled data displaying some strange results? And even then I don't see why the actual figures are that important.

Im only after a hand on understanding if heavy metals can have activating effects at low concentrations as well as the expected inhibitory effects on beta-galactosidase. Like I've already said I'm struggling to find anything in the literature that agree's with my data. However I have emailed my lecturer and he has hinted towards my data not being wrong after all. 

I'm probably just gonna roll with zinc possibly acting as a substitute for magnesium at low concentrations as it seems plausible to me.