catalyze specific chemical reactions. E + S ↔ E-S Complex ↔ E + P. Factors
Affecting Enzyme. Activity. • Concentration of Enzyme. • Concentration of
Substrate.
Enzymes Enzymes Lab Section 2.4
• Protein catalysts • Have complex 3-D structures • Pockets act as active sites – catalyze specific chemical reactions
E + S ↔ E-S Complex ↔ E + P
Factors Affecting Enzyme Activity • Concentration of Enzyme • Concentration of Substrate • Concentration of Cofactors / Coenzymes • Temperature • pH
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]…
Measuring Enzyme Concentration Using Beer’s Law • Measure [enzyme] indirectly via reaction rates – Enzyme activity (reaction rate) α [enzyme]
• During the reaction, substrate → product – Rate = how much product is formed per unit time – So, [Enzyme] α ∆[Product]/time
International Units (U) • Measurement of enzymatic activity (U) – Quantity of enzyme needed to convert 1 µmol substrate into product in 1 minute
• Measure of enzymatic function, indirectly enzyme concentration • Often expressed as concentration (U/L) • Determined by examining ∆[product] / min
• Therefore: A/time α [enzyme]
1
Alkaline Phosphatase (ALP) • Phosphatase
Procedures • Timing is critical once the reaction has begun!
– 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
– 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)
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
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)
