Calculator
Use the responsive grid below. Large screens show three columns, medium screens show two, and mobile screens show one.
Example Data Table
These rows illustrate how different assay setups change calculated activity.
| Assay | Sample start | Sample end | Blank start | Blank end | Time | ε | Net slope | Activity | Specific activity |
|---|---|---|---|---|---|---|---|---|---|
| NADH-linked assay | 0.120 | 0.420 | 0.010 | 0.030 | 3.00 min | 6,220 | 0.093333 | 0.450161 U/mL | 0.180064 U/mg |
| Oxidation assay | 0.300 | 0.180 | 0.015 | 0.009 | 2.00 min | 6,220 | -0.057000 | 0.366559 U/mL | 0.203644 U/mg |
| Chromogenic assay | 0.050 | 0.260 | 0.005 | 0.011 | 4.00 min | 18,000 | 0.051000 | 0.141667 U/mL | 0.044271 U/mg |
Formula Used
1. Sample slope: (Aend − Astart) ÷ time
2. Blank slope: (Blank end − Blank start) ÷ time
3. Corrected slope: Sample slope − Blank slope
4. Concentration rate: |Corrected slope| ÷ (ε × path length)
5. Cuvette rate: Concentration rate × total volume in liters × 106
6. Activity: Cuvette rate × dilution factor ÷ enzyme volume
7. Specific activity: Activity ÷ protein concentration
This workflow applies the Beer–Lambert relationship and converts absorbance change into enzyme units, usually defined as micromoles converted per minute.
How to Use This Calculator
- Enter the assay name for easier result tracking.
- Provide sample absorbance values at the start and end.
- Provide blank absorbance values for background correction.
- Enter the measurement interval in minutes.
- Type the extinction coefficient for your product or substrate.
- Enter cuvette path length, total reaction volume, and enzyme volume.
- Add the dilution factor used before the assay.
- Optionally enter protein concentration to calculate specific activity.
- Press the calculate button to show results above the form.
- Use the export buttons to save result tables as CSV or PDF.
Frequently Asked Questions
1. What does the extinction coefficient represent?
It expresses how strongly a compound absorbs light at a chosen wavelength. Higher values mean a small concentration change creates a larger absorbance change.
2. Why should I include blank readings?
Blank readings remove background drift from buffers, cuvettes, and instrument noise. This gives a corrected slope that better reflects the enzyme-driven reaction.
3. Why does the calculator use the absolute net slope?
Some assays increase absorbance, while others decrease it. Using the absolute value keeps activity positive while still showing the actual reaction direction separately.
4. What unit does U/mL mean?
U/mL means enzyme units per milliliter of enzyme sample. One unit commonly equals one micromole of substrate converted each minute.
5. When is specific activity useful?
Specific activity is helpful when comparing purity or enzyme enrichment. It normalizes activity to protein concentration and reports performance per milligram of protein.
6. What path length should I use?
Use the effective optical path length for your cuvette or microplate well. Standard cuvettes are often one centimeter, but microplates usually need correction.
7. Can I use this for decreasing absorbance assays?
Yes. Decreasing absorbance is common in oxidation or substrate depletion assays. The calculator still determines activity and labels the measured direction.
8. Why might my activity seem unusually high?
Check dilution factor, volume units, path length, and extinction coefficient first. Small entry mistakes can strongly affect the final activity value.