Principles of Enzyme assays

Question 1

Why measure enzymes in clinical medicine?

Answer 1

• diagnosis of genetic disorders
• patterns of serum enzyme activity may identify tissue source and likely diagnosis
• e.g. elevated levels of particular enzymes found in blood samples - can detect which organ isnt working properly
• loss of secreted enzymes into GI tract e.g. pancreatic disease

Question 2

Michaelis-menton curve

Answer 2

• relating velocity (rate) to concentration of substance

- the Km is the concentration of substrate at which 50% max velocity (Vmax) is achieved

Question 3

Conditions for activity measurements

Answer 3

• temp - (Q10 = relative reaction rates at two different temps different by 10 degrees) - for most systems rate doubles with a 10 degree increase
• pH optimum (can vary from 1.5-10)
• precise substrate concentration and ionic strength of buffers

Question 4

Units of enzyme activity

Answer 4

• International unit (IU) - one unit catalyses the conversion of 1umol of substrate in 1 min
• Katal - 1 katal converts 1 mol substrate/seconds
• 1kat = 60mol/min
• 1IU = 1umol/min

Question 5

Practical considerations

Answer 5

• enzyme purity and nature
• enzyme stability
• quality of substrates, solvents and buffers
• assay mixture
• mixing to start reaction

Question 6

Some methods of continuously monitoring enzyme catalysed reactions

Answer 6

• absorbance
• fluorescence - substrate or product may have normal level of fluorescence, otherwise make it fluoresce by putting in energy
• optical rotation - stereoisomer/chiral centre
• conductivity
• viscosity
• radiochemical - measuring radioactivity

Question 7

Spectophotmetry

Answer 7

• if substrate or product absorbs light, reaction can be followed by change in absorbance
• Absorbance is related to concentration of substance
• A = ecl
• e = molar extinction coefficient, c = molar concentration and l = light path length

Question 8

What do we do to avoid analytical problems?

Answer 8

• standardisation of assays
• quality control - must know coefficient of variation (CV)
• (SD of control values / mean of the control values) x 100

Question 9

Lactate dehydrogenase

Answer 9

CH3COCOO- + NADH CH3CHOHCOO- + NAD+

• NAD has little or no absorbance at 340nm, so can measure drop in absorbance of NADH
• when you have a heart attack, tissue is destroted, lactate dehydrogenase is released into the blood

Question 10

Alkaline phosphatase (ALP)

Answer 10

• High levels in blood are indicative of liver damage or bone disease

Question 11

Enzymes as analytical reagents

Answer 11

• Glucose + ATP -> G6P + ADP (hexokinase)

- G6P + NAD -> 6-phosphogluconate + NADH + H+ (G-6-PDH)

Question 12

Clinically important enzymes

Answer 12

• amylase (pancreatitis)
• alkaline phosphatase (liver/bone)
• alanine aminotransferase (liver)
• aspartate aminotransferase (liver, infraction)
• lactate dehydrogenase (haemolysis, lymphoma)
• creatine kinase (skeletal disorders)