Enzymes assays Flashcards
Many diseases that cause tissue damage / injury result in ——release of intracellular enzymes into the —-.
inc.
plasma
What is Macro-amylase-emia?
Macromolecules of amylase (aggregates with Ig - LDH, ALP, CK) = rate of clearance reduced = Amylase Higher than normal
Which of the following enzymes is normally detected in urine, and why?
Amylase bc the other enzymes are too large to cross the glomerulus
pH range of blood
7.35-7.45
During purification process of enzymes, the assay must be __ because the ___ of most enzymes are very low.
highly sensitive
specific activities
When measuring enzyme activity, you add acidic 2,4-dinitrophenylhydrazine, why?
(after 30min of assay) Denature enzymes = stop activity = can be measured at 505nm = detect end point
Equation to finding [ ] of test =
[ Abs(test) / Abs(Std) ] x C(std) *NOTE: equ. comes from A(test) C(test) \_\_\_\_\_ = \_\_\_\_\_ A(Std) C(Std)
define product inhibition
too much product can inhibit the enzyme bc product can bind again to active site & prevent new substrate reacting
Describe coupled enzyme assays(*cont)
if reactant or product is colourless*
State two factors that can raise plasma ALP activity and they are not diseases? Explain your answer.
- Age, sex, race/ethinicity, physiological conditions
* 3rd trimester pregnancy: 30 – 125 U/L
in protein electrophoresis what proteins are found on the gamma region (near the anode)?
- CRP
- Ig*
- Fibrinogen
in protein electrophoresis what proteins are found on the beta region?
- transferrin
- LDL
in protein electrophoresis what proteins are found on the alpha-2 region?
- Haptoglobin
- HDL
Describe the order of zones in protein electrophoresis from cathode to anode.
(neg) Albumin - alpha1 - alpha2 - beta - gamma (pos)
Enzymes can give information on
- Presence of disease
- Which organ(s) is diseased
- Extent / severity of the disease – more cell damaged means more leaked enzymes … severe disease
- Time course of the disease
Discus with examples, why the enzymes’ level could be abnormally higher or lower than the normal in blood?
- Acute: cell damage = Higher e.g. acute pancreatitis
- Chronic: reduce synthesis = lower e.g. late chronic pancreatitis
Prostatic cancer elevates serum levels of ——-.(enzyme)
Acid phosphatase
Difference b/w the units Katal (K) and International Unit (U)
- K: amount which transforms 1 mole substrate/sec
* U: amount which transforms 1 µmole substrate/min
Which of the following organs/tissues is rich of the following enzymes, CK, AST, ALT, aldolase and LDH
muscle
What’s Paget’s disease
- bone disease
- ALP levels are 10x greater than upper limit of normal
A useful enzyme assay must meet the following criteria
(a) absolute specificity (specific to one or more known substrates)
(b) high sensitivity
(c) high precision & accuracy
(d) convenience
Beer lambert’s law
A=E.C.L
The enzymatic reaction for glucose assay in presence of glucose oxidase is detected using which chromogen/light absorbing compound?
Quinoneimine
The enzymatic reaction for glucose assay in presence of__ is detected using __ (chromagen compound)
hexokinase
NADH
The enzymatic assay of__ is carried out using ___. The produced ammonia is further reacted with different compounds including sodium nitroprusside to produce the chromogen compound —-
urea
urease
Phenol
The enzymatic reaction for cholesterol assay in presence of __ esterase is detected using __
cholesterol
Quinoneimine
The enzymatic reaction for ___ assay in presence of lipases is detected using ___
triglyceride
Quinoneimine
To improve the specificity of CK-MB 2 things are determined
- Measure mass instead of activity = (mass/activity) x100
- Measure activity of CK-MB as a ratio w/ CK = (CK-MB activity / CK activity) x100
*If CK-MB activity is:
>5 = myocardial infarction
<5 = damage in skeletal muscles
Substrate, product, activator, pH of alkaline phosphatase
- substrate: p-nitrophenol phosphate
- product: p-nitrophenol
- Activator: magnesium sulfate (accelerators)
- pH: ~9
Michealis Menten rule & note
• Vmax is proportional to the amount of enzyme added
PS. [S]»Km: zero-order kinetics and v independent of [S]
What does it mean to have a substrate-enzyme reaction with low Km?
Lo Km = Hi affinity = lo [S] required to achieve Vmax
What does it mean to have a substrate-enzyme reaction with Hi Km?
Hi Km = Lo affinity = Hi [S] required to achieve Vmax
difference b/w Lineweaver burk plot vs Hanse plot in terms of the graph
- LWBP: y= 1/[v], x=1/[S], m= km/Vmax
* HP: y= -km, x= S, m= 1/Vmax
