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Understanding Enzymes: Catalysis, Measurement, and Alkaline Phosphatase Activity

Enzymes are complex protein catalysts with specific 3-D structures that facilitate chemical reactions by creating active sites where substrates transform into products. Factors like enzyme and substrate concentration, temperature, and pH influence enzyme activity. Measurement of enzyme concentration can be performed using Beer’s Law, relating absorbance to product formation. Alkaline Phosphatase (ALP) is a key enzyme in diagnosing conditions such as liver disease, with its activity measured through absorbance changes over time. Understanding these concepts is crucial for biochemical analysis.

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Understanding Enzymes: Catalysis, Measurement, and Alkaline Phosphatase Activity

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  1. Enzymes Lab Section 2.4

  2. Enzymes • Protein catalysts • Have complex 3-D structures • Pockets act as active sites • catalyze specific chemical reactions Fig 2.8, p. 7 E + S  E-S Complex  E + P

  3. Factors Affecting Enzyme Activity • Concentration of Enzyme • Concentration of Substrate • Concentration of Cofactors / Coenzymes • Temperature • pH See p. 7

  4. Measuring Enzyme Concentration Using Beer’s Law • Measure [enzyme] indirectly via reaction rates • Enzyme activity (reaction rate) a [enzyme] • During the reaction, substrate → product • Rate = how much product is formed per unit time • So, [Enzyme] αΔ[Product]/time

  5. Measuring Enzyme Concentration Using Beer’s Law • If product formed absorbs light… • [product] α A • As [product] changes, A changes proportionally • Since [product]/time α A/time and [product]/time α [enzyme]… • Therefore: A/time α [enzyme]

  6. International Units (U) • Measurement of enzymatic activity (U) • Quantity of enzyme needed to convert 1 mmol substrate into product in 1 minute • Measure of enzymatic function, indirectly enzyme concentration • Often expressed as concentration (U/L) • Determined by examining D[product] / min

  7. Alkaline Phosphatase (ALP) • Phosphatase • Enzyme that removes phosphate groups from proteins • Normally low levels of ALP in blood plasma • Elevated levels indicate pathology • Liver and bone disease, Hodgkin’s disease, congestive heart failure, hyperparathyroidism, intestinal disease, etc. • Enzyme activity measured by reacting with substrate to form light-absorbing product • p-nitrophenol phosphate + H2O p-nitrophenol + hydrogen phosphate

  8. Procedures • Timing is critical once the reaction has begun! • Have your spec already zeroed with the blank before you start the reaction. • use 2 samples: • BLANK (3.0 ml of reagent and 0.1 ml distilled water) • BLOOD (3.0 ml of reagent and 0.1 ml serum) • Place in test tube in 30 C bath for 1 min

  9. Procedures • Remove tube, dry outside, place in spec, and read absorbance • Replace in water bath • Remove, dry and read absorbance at 2 min • Repeat to measure absorbance at 3 min, 4 min, and 5 min

  10. Determining ALP Activity Determine average change in absorbance per minute (A5min – A1min)/4 = A/min Determine alkaline phosphatase activity A/min x TV (ml) x 1000 (ml/L) = activity (U/L) 18.45 x LP x SV (ml)

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