Title: Assay of Alkaline Phosphatase in Human Serum
Aim: To determine the activity of alkaline phosphatase (ALP) in human serum using a spectrophotometric method.
Introduction:
Alkaline phosphatase (ALP) is an enzyme found in various tissues, including the liver, bones, and kidneys. It plays a crucial role in dephosphorylation reactions, removing phosphate groups from molecules. Measuring ALP activity in human serum is clinically significant, as elevated levels may indicate liver or bone disorders. This assay is widely used in medical diagnostics and research.
Theory:
ALP catalyzes the hydrolysis of phosphate esters in an alkaline environment, releasing inorganic phosphate and alcohol. The enzyme activity can be measured by using p-nitrophenyl phosphate (pNPP) as a substrate. ALP hydrolyzes pNPP to p-nitrophenol (pNP), which produces a yellow color in an alkaline medium. The intensity of the yellow color, measured at 405 nm using a spectrophotometer, correlates with ALP activity in the serum sample.
Principle:
ALP hydrolyzes p-nitrophenyl phosphate (pNPP) to produce p-nitrophenol (pNP) and phosphate:
p-Nitrophenyl phosphate + H₂O → p-Nitrophenol + Phosphate
The yellow color intensity of p-nitrophenol is directly proportional to the enzyme activity and is measured at 405 nm.
Requirements:
Human serum sample
Alkaline phosphatase reagent (pNPP substrate solution)
Buffer solution (pH 10)
Standard ALP solution (for calibration curve)
Test tubes
Pipettes and micropipettes
Water bath (37°C)
Spectrophotometer
Stopwatch
Procedure:
Prepare the pNPP substrate solution in alkaline buffer (pH 10).
Prepare a standard ALP solution for calibration.
Label test tubes for blank, standard, and serum samples.
Add 1 mL of pNPP substrate solution to each test tube.
Add 0.1 mL of serum sample to the test tube labeled “serum.”
Add 0.1 mL of standard ALP solution to the test tube labeled “standard.”
Add 0.1 mL of buffer to the test tube labeled “blank.”
Incubate all test tubes at 37°C for 30 minutes.
Stop the reaction by adding 1 mL of NaOH (0.1M) to each tube.
Observe the yellow color development in the test tubes and Measure absorbance at 405 nm using a spectrophotometer.
Result:
Higher absorbance at 405 nm indicates higher ALP activity in the serum sample.
The enzyme activity can be calculated using a calibration curve or the standard ALP formula.
Conclusion:
The assay successfully determined the activity of alkaline phosphatase in human serum. The intensity of yellow color formation is directly proportional to ALP concentration, making this method useful for clinical diagnostics and enzyme activity studies.