[biomed777]

hi there am new to this forum, so nice to meet you all!

we did an experiment last week on the spectrophotometric determination of lactate dehydrogenate activity

we had 2 buffers: one containing 1ml of 50mM TRI/ 1mM EDTA/ 20mM mercaptoethanol/ 5mM MgCl2 the buffer was pH 7.4


the second buffer cntained 1mM of 50mM TRIS/ 1mM EDTA/ 5mM MgCl2 the buffer was pH 7.8

the tissue extract was mixed with buffer one but we set up the spectrophotometer to zero with buffer two.

can someone please shed me some light and help me on why we used the second buffer? is it something to do with the pH??

Many thanks in advance

[Arkcon]

They seem to be exactly the same, except for one reagent.  Do you know anything about the properties of that reagent that would make you leave it out?

[biomed777]

no, i really dont get it, the only difference is that the other buffer contains 20mM of mercaptoethanol

[Babcock_Hall]

What do you know about the chemical and spectroscopic properties of 2-mercaptoethanol?  What wavelength of light were you using in this experiment?

[biomed777]

hi there and thanks for your reply it was 340nm wavelength. now i read that mercaptoethanol denatures proteins? useful for protein denaturation? again does not make any sense to me

[Babcock_Hall]

340 nm is the wavelength maximum for NADH.  2-ME is a reducing agent.  It would denature a protein that had disulfide bonds, but it would keep a protein that had reduced cysteine residues from denaturing.  You might want to look into the spectroscopic properties of the oxidized form of 2-ME.