I have a real problem in calculating the activity of tartrate resistant acid phosphatase by a kinetic assay illustrated in a paper in the following link:
and this is a part of the paper:
2,6-Dichloro-4-acetylphenyl phosphate (DCAPP) was obtained from Nitto Boseki Co. Ltd. Sodium L-(+)-tartrate and heparin were obtained from Sigma, and polybrene was obtained from Aldrich. Carboxymethyl-agarose (CM-Sepharose) was obtained from Pharmacia Fine Chemicals. All other analytical grade chemicals were purchased from Wako Pure Chemical Co.
The reaction solutions we reported previously (12) were used with some modification. Briefly, buffer solution I consisted of 150 mmol/L 2-(-hydroxy-3-morpholino) propanesulfonic acid, 60 mmol/L sodium L-(+)-tartrate, 23 kIU/L heparin, and 5 g/L bovine albumin (pH 6.6). Buffer solution II consisted of buffer solution I plus sodium fluoride (45 mmol/L). The substrate solution consisted of 45 mmol/L DCAPP and 50 mmol/L Tris (pH 4.0).
Blood samples collected from 516 apparently healthy Japanese subjects, ages 5–79 years (210 males and 306 females), by clean venipuncture were allowed to clot at room temperature for 2–4 h and centrifuged at 1000g for 10 min at room temperature. Serum thus separated was transferred into 1.5-mL tubes and stored at -80 °C until use (within 6 months).
Bone extract samples were prepared as described previously (12). In brief, bovine tibia after the removal of soft tissue was cut into small cubes. The marrow and blood were removed, and the bone was ground to powder and homogenized in a solution containing Triton X-100, potassium chloride, phenylmethylsulfonyl fluoride, benzamidine, and aminocaproic acid. The extract was collected by centrifugation at 10 000g for 20 min at 4 °C and stored at -80 °C until use.
TrACP activity was measured using a centrifugal automatic analyzer (Cobas Fara; Hoffmann-La Roche). Briefly, 150 µL of buffer solution I was added to 15 µL of sample, and the mixture was incubated at 37 °C for 5 min. The enzyme-substrate reaction was initiated by the addition of 60 µL of substrate solution. The change in absorbance at 340 nm was monitored at 20-s intervals for 5 min. The millimolar absorptivity of the hydrolysis product (2,6-dichloro-4-acetylphenol) is 21.49 L · mmol-1 · cm-1 at 340 nm. One IUB unit (1 U) of TrACP activity is defined as 1 µmol of DCAPP hydrolyzed per minute at 37 °C in the presence of 40 mmol/L sodium L-(+)-tartrate and 23 kIU/L heparin at pH 6.6. Tartrate-resistant fluoride-resistant acid phosphatase activity was assayed using buffer solution II instead of buffer solution I. Band 5b TrACP activity was estimated by subtracting tartrate-resistant fluoride-resistant acid phosphatase activity from TrACP activity.
The italic part is the procedure of the assay... I need to know how to calculate the activity using many absorbances at 20 second intervals till 5 min... I couldn't get it simply...
I hope if there is anyone can help or direct me, please tell me.....