[rycharles]

In lab we've performed affinity and gel filtration chromatography on our pig heart sample. We used Cibacron Blue Sepharose, which attracts proteins with nucleotide cofactors, in our affinity column.  In gel filtration chromatography, we ran the sample through HPLC.

The purpose of the series of experiments is to determine LDH.  HPLC showed proteins at different peaks.  We then assayed these fractions and found LDH activity in one of the peaks.  My question is, what other proteins or non-proteins are causing the other peaks in HPLC, but not showing activity when being assayed (the reaction cocktail consists of CAPS buffer, NAD+, lactate, and the protein sample)?

[Babcock_Hall]

You are supposed to show an attempt first, before we give you help.  Can you think of any possibilities?  Fortunately, the seed of a decent answer is found within your message.

[rycharles]

Well from the affinity chromatography, we've excluded proteins that do not use nucleotide cofactors.  Since cibacron blue is similar to a nucleotide cofactor, we can have proteins that utilize other nucleotide cofactors-not just NAD+.  Other nucleotide cofactors could be coenzyme A, FAD, FMN, NADP+... So we could be seeing proteins that utilize the other cofactors?  Is this why we do not see activity when we assay the other fraction samples?

As for the gel filtration chromatography, I don't know what the range of size would be.  But I do know that LDH (a tetramer with 4 subunits) is 140,000 daltons in size.

[Babcock_Hall]

IIRC each monomer of lactate dehydrogenase is 140,000; therefore, the tetramer is larger than that.  Your point about CoA and NADP is quite reasonable.  Also LDH is not the only enzyme that uses NAD as its coenzyme.  Other examples would include malate dehydrogenase and alcohol dehydrogenase, etc.  They might not use lactate as a substrate at all, or they might use it at a very low rate, which could escape detection.