Please look at the following below links which may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1144952/pdf/biochemj00303-0063.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1147062/pdf/biochemj00274-0194.pdf

Thanks

To calculate the initial rate of the reaction, you must be able to identify a linear portion of the progress curve at the start and measure its slope. In the case of your curve B, it is problematic because the curve increased sharply in the first 30 seconds, then transitioned to a shallower, nonlinear increase. It is unclear whether the first 30-seconds represents the initial rate or is an artifact, and the later part of the curve has no linear portion.

The reaction may have occurred too quickly, so that you missed the initial rate by the time you started collecting data. This can be solved by lowering the enzyme concentration.

The first part of the curve may be a case of burst kinetics, which can be observed if the enzyme concentration is high enough. This can be eliminated by lowering the enzyme concentration.

If the DTNB concentration is too low, the curve may represent the rate of the product detection reaction instead of the product production reaction. This problem can be solved by increasing the DTNB concentration.

The first part of the curve may represent the reaction of DTNB with cysteine residues in the enzyme, or thiol-containing reducing agent carried over with the enzyme from the storage buffer. This effect can be reduced by lowering the enzyme concentration.

The later part of the curve may represent substrate depletion or product inhibition, or inhibition of the enzyme by DTNB.

I suggest you repeat this measurement using several lower enzyme concentrations to try to understand the progress curve better, and to see if you obtain a linear progress curve. You may also have to increase the substrate concentration to reduce substrate depletion and product inhibition. Also, consider running this reaction discontinuously, to avoid enzyme inhibition by DTNB. For that, you need a quencher to stop the reaction when the DTNB is added.

Adam B Shapiro , Thank you very much for your recommendations.

I DID repeat the measurement using different enzyme concentration, but I'm still seeing a sharp increase in the first 0-30sec time range... Just like other papers, I used 130uM DTNB, 130uM Ac-CoA, 10uM AcacCoA in 100uL in 96-well, and the absorbance values were measured every 5s. Before the measurement, the plate was mixed for 2s.

I do believe that the first part may be the reaction of DTNB w/ cyteine of the protein since I have eliminated reducing agent of the enzyme storage buffer...

Should i recondition the concentration of the substrates../?

Considering that the size of the initial rapid deflection increases with the amount of enzyme added, and all the other components are constant, I agree that the deflection is most likely caused by DTNB reacting with the enzyme. You can eliminate that part of the curve by premixing the DTNB with the enzyme, before adding the substrates.

Another possible problem is that there might be a fast reaction of DTNB with any contaminating CoA in the substrates. To eliminate that, you could also premix the substrates with DTNB.

With both of those unwanted reactions occurring before the enzyme and substrates are mixed, perhaps you will finally get a linear progress curve. However, with only 10 µM AcacCoA, the size of the absorbance increase will be very small. I think you will have to increase the AcacCoA concentration to get a measurable linear absorbance increase. Here are some numbers to explain why:

The extinction coefficient change when DTNB reacts with thiol is 13,600 M-1cm-1 at 412 nm. For a 100 µl reaction in a 96-well plate, the path length is roughly 0.5 cm. Converting 1 µM acyl-CoA to CoA will therefore result in an absorbance change of about 0.0065, just over 1 minor division on your graph. Since that represents 10% of the AcacCoA concentration, that is likely to be the full extent of the initial rate part of the curve. It could be a bit more if the Km for AcacCoA is less than 1 µM.

Adam B Shapiro yes, this time, I used less enzyme, and used 10uM of acac-coA (other conditions were the same)... but still got a sharp increase in the first 0-30secs... which confuses me so much. ;(

The reason why I am limiting the concentration of Acac-coa is because the DTNB solution reacts with the substrates faster... (As soon as I drop acac-coa into the solution, the assay buffer immediately turns yellow).

For this assay, I first added solution 2 (enzyme in buffer solution + Ac-ac-CoA) into a microplate, and right after adding solution 1 (Ac-Coa premixed in DTNB solution/buffer), I let the microplate reader to shake for 5 seconds, and started reading the absorbance values.

It seems like the reference below has determined the the rates by measuring the o.d. values after 3mins of incubation of solution 1+2....

Article A visible wavelength spectrophotometric assay suitable for h...

It sounds like a big part of your problem is that the AcacCoA is of low purity and contains a large amount of free sulfhydryl, probably CoA. It would probably be worth spending some time on trying to purify the substrate to avoid this high background. Then you would be able to use a high enough concentration to get a large enough signal from the reaction.

Waiting 3 minutes to read the rate is an option, if (1) you are sure that the rate at that time represents the initial rate of the reaction, and (2) if the progress curve is linear. With such a small AcacCoA concentration, much of which may not actually be AcacCoA, however, it is unlikely that there is any point at which you can measure the initial rate. There just isn't enough AcacCoA in the reaction.

If you think that you are looking at a hyperbolic time course, you could perform a progress curve analysis. The idea is that each progress curve contains info on the enzyme rate at an infinite number of substrate concentrations. See for example doi:10.1016/0076-6879(79)63010-0. However, looking at your data, I'd say that there is either an offset, or a convolution of two different velocities.

Sewon, I have looked to the paper you cited just a bit. In their Fig.1, the inset shows the initial velocity determination over a 4 min period, exhibiting linear changes but very small absorbance increase at 412 nm - so their measurements are resembling your "A" measurement in the very first graph you provided. It looks like your conditions are in parallel with the original paper's conditions at this measurement. However, Adam and you have been discussing on your "B" measurement, which is obviously much different from "A". Whatever is the reason for this difference, I guess you should either stick to the conditions in exp "A", or figure out on a step-by-step manner why your exp "B" is so much different, starting from conditions in exp "A".

On the other hand, I agree with Adam's explanations, also Engelbert's paper is useful to get some hints on the problem with exp "B". Looking at the paper Engelbert cited, there is a possibility that your enzyme looses activity/stability (but other reasons might explain the decline starting at 500 sec).

Finally, again referencing the original paper describing the method, you could utilize the first 4-5 min of the measurement to estimate initial velocity, supposing that the reason responsible for the decline around 10 min and later not affecting the first few minutes. Nevertheless, I suggest troubleshooting the conditions...

Karoly Liliom The first graph of Measurement A is just a basal state (without the enzyme) that contains only DTNB and the substrates. Measurement B is the one that actually represents the progress curve. I always measure the basal because DTNB cross-reacts with the substrates.

All the other references (regarding measuring the initial velocity of HMGCS) has followed MM kinetics which means.. the progress curve must have been linear...

If change in conc. of enzyme or substrate is showing less variation, then you should try to change pH of buffer solution or temperature. Bcz these two parameters can slow down reaction and thus easy to monitor the reaction.