You want the mol/L. The activity is the rate of conversion of NADH to NAD+ at 340 nm.

That gives the conversion of the coenzyme in the forward reaction - pyr to lactate - with the oxidation of NADH. Isn't that actually per mol of LDH.

I have been away from this for 4 decades. However, I published related work in Methods in Enzymology with Johannes Everse in the late or mid 1970's.

The answer to the question is more complicated because the enzyme is a tetramer with 4 subunits, and five species: H4, H3M, H2M2, HM3, M4. The H-type enzyme is regulatory, but not the M-type. If you used M4, there is no inhibition by an abortive ternary complex. If you used H4 from heart, you would see inhibition by the abortive complex in an Aminco-Morrow stop flow in 500 msec. I have saved data, but would have to look for it. The study was done with purified enzyme from human autopsy.

The Km for both types is the same. The difference in reaction is a result of:

LDH + NAD+ (generated in the forward reaction) + pyruvate

as activation of enzymes increae upon ageing, there is no simple relationship between activity and mass; moreover, there are 2 different subunits (H and M) which build up the various isoenzymes, LDH-1 (M4) will have a preferencve for beta hydroxybutyric acid)

I assume this assay is for a clinical laboratory, so you already know that LDH is measured by activity. If the patient has high [LDH], the relative amounts of LDH isozymes are determined by electrophoresis to determine the organ system that is affected.

This makes no sense to me. beta hydroxybutyric acid is about 2/3 of the composition of ketone bodies. Ketoacidosis does occur when pyruvate entry into the LDH reaction is blocked, so that thee is no buildup of lactic acid, and ketones are generated through the TCA cycle, which I haven't reviewed in over 40 years. I am open to correction. But this statement about age related increased activation of enzymes is an unproved hypothesis in general principle, although the interaction or redirection of metabolic pathways is extremely important. I don't know of any work on this as an epigenetic event. I think that the accumulation of substrates would only tie in with a back-accumulation between reactions. This also might tie in with the separate reverse reaction of lactate conversion, and with malate, aspartate and fumarase.

I pointed out that the purified H4 is regulated by the formation of an abortive complex that forms as the forward reaction proceeds and the inhibition can be seen at 500 msec on the Aminco Morrow. The Km of the LDH reaction is the fingerprint. You can do the reaction with alternative substrates that hjave a different Km. The mechanism of the malate dehydrogenase reaction is basically the same, but with the c- and m-type MDH you have a ternary complex occurring in both. However, as Warburg thought that cancer involves impaired mitochondrial function (a Pasteur effect under aerobic conditions) (one study identified a block at fumarase in the TCA cycle) we found that in minimal-deviation and fast-growing hepatomas from Herschel Sidranski's lab the m-MDH was not the problem, but the cytoplasmic MDH continued to increase its activity of NADH oxidation with increasing oxaloacetate without the expected flattening of the curve. I was unable to continue the studies at another level after receiving approval without funding.

All you needed is to measure the concentration of total protein in your sample using Lowry or Bradford determination test which are supplied as commercial kits. they usualy report the total protein concentration as micro mg/ml. In next step, you divide the measured activity of LDH in sample in case of (mol/L) to the total protein. Now you have the LDH specific activity that is expressed as mol/g and is comparable to other samples of yours with no need of normalization.

good luck

It is possible to approximate human LDH concentration from activity. What you need is to do several repetition of your experiments to validate your results statistically.

I'm afraid you cannot use the kit assay for scientific validity. NAD is cheap, so the kit runs the reverse reaction - NAD to NADH. That is what industry considered - the reagent cost. As you well know, other enzymes are measured used coupled reactions, and following the OD at 340 nm. In the clinical setting, you have to run controls, and you have to also participate in Proficiency Testing.

Of course, with Eliza - you are measuring the protein molecule. I don't know what the effect the multiple tetrameric forms would have on the assay. Today, one might use affinity chromatography.

You are running a standard assay for the reduction of NAD+ with lactate as substrate. You are comparing this with an Elisa. Am I getting it right? Is it your intent to subsequently use the LDH immobilized on the bead as the reporter?

The Elisa is very sensitive. Perhaps more than the backward or the forward reaction. But I can see why you would want to see the increase in OD as the reaction develops, rather than the forward reaction, which is actually more sensitive. You haven't state whether you are using a purified muscle enzyme.

This would be possible provided you have the initial rate V0, however you cannot obtain V0 unless you assay the kinetics of the samples of LDH. Given the KM and KCAT constants for LDH as well as the substrate concentration as well as the initial rate and according to Michaelis - Menten equation you can obtain the enzyme concentration. This method is theoretical I didn't find any standardized protocol to verify it... So I don't think you can use this method to quantify enzyme amounts..

Thanks for your answers and the discussion everyone. I am actually intending to look at total LDH levels of all isoforms. To clarify, I am using clinical samples as well.

Larry I haven't considered using affinity chromatography. That is a great Idea; I will definitely look into it.

You might be able to contact Prof. Emeritus, Johannes Everse at Texas Tech. He synthesized adducts of the coenzyme 40 years ago, and he also did some work with early affinity chromatography. He would have to go back in records to find methods of use to you.

If you could put your heterogeneous sample on a column to which the enzyme specifically attaches, wash through the column, and then elute with a specific reagent, it would save you months of work.