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Enzymes

Enzymes. Lab Section 2.4. 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. Factors Affecting Enzyme Activity. Concentration of Enzyme Concentration of Substrate

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Enzymes

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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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