The activity of an enzyme is measured in katal (1 kat = 1 mol/s), the non-SI enzyme unit U (µmol/min, 1 U = 16.7 nkat) should no longer be used.

If you incubate a substrate with an enzyme, the concentration of the substrate will decrease, that of the product will increase over time. Under appropriate conditions, these changes are linear, and the enzyme activity is the slope of that line.

Thus, the procedure is always the same: Measure the substrate or product concentration either continuously or at several time points, plot the data points over time and check for linearity, then do a linear regression to determine the slope.

Never, ever use a single time point to do that determination, you do not know whether the reaction rate was linear, and you cannot determine the standard deviation of the slope.

Up to which point it depends on the method Reitman & Frankel(1957)? This is something which matters. Read thoroughly the protocol, there might be times where you must not mix. The units may be ordinary or not, but see if there is anything else.

check below link might be useful

https://www.aacc.org/~/media/files/meetings-and-events/resources-from-past-events/conferences/2015/profprac/slides-and-handouts/laboratory_calculations_handouts_jones.pdf?la=en

1 enzyme unit = the enzyme activity that catalyses the conversion of 1 µmol substrate into product in 1 minute

Data Analysis Calculation of Results To determine the slope of the pyruvate standard curve, use the t=35 min measurements. Subtract the blank value (#4) from the standard values and plot the ∆OD or ∆F against standard concentrations. Determine the slope by linear regression. To determine the PK activity in the samples, first subtract the OD5min from OD35min or F5min from F35min for each sample and the 0 µM Pyruvate Standard (Blank). The PK activity can then be computed as follows: SAMPLE BLANK -1 R - R [PK]= x n (U/L) Slope (µM ) x t ∆ ∆ where ∆RSAMPLE and ∆RBLANK are the changes over time in the optical density or the fluorescence intensity readings of the Sample and Blank, respectively. t is the time of reaction (30 min). n is the sample dilution factor (e.g. n = 4 for serum). Note: if the calculated PK activity is higher than 50 U/L for the colorimetric assay or higher than 2 U/L for the fluorimetric assay, dilute sample in dH2O and repeat assay. Multiply result by the dilution factor n. Unit definition: 1 unit of PK will generate 1 µmole of pyruvate and 1 µmole ATP from PEP and ADP per minute at 25 °C at pH 7.5.

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