2. Enzymes Lab Section 2.4

3. Enzymes Protein catalysts Have complex 3-D structures Pockets act as active sites –catalyze specific chemical reactions E + S  E-S Complex  E + P

4. Factors Affecting Enzyme Activity Concentration of Enzyme Concentration of Substrate Concentration of Cofactors / Coenzymes Temperature pH

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

6. 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]

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

8. 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 + H 2 O  p-nitrophenol + hydrogen phosphate

9. 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

10. 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

11. Determining ALP Activity Determine average change in absorbance per minute (A 5min – A 1min )/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)