CC LEC 2 - Enzymes Flashcards
most enzymes react at pH __but some are active in wider pH ranges
7.0- 8.0,
ALP –
pH
pH 9.0-10.0
Gastric enzyme – pH
pH 6.0
For each ?°C increase in temp. reaction rate doubles
until the protein is denatured
10
– should always be provided in excess so that the extent of the reaction does not depend on its concentration
Cofactors
What are measured in measurement of enz activity? (4)
INC in product
DEC in substrate
DEC in coenzyme
INC in altered coENZ
In coupled ENZ assay,
-what catalyzes its reaction, product formed becomes a substrate of intermediate reaction.
enzyme under analysis
In coupled ENZ assay,
-what produces an intermediate product that will be the substrate for the final reaction
Intermediate auxiliary enzyme
What absorbs light at 340 nm while NAD does not)
NADH
Usually involves the conversion of NAD to NADH or vice versa [what enzyme type in coupled ENZ)
indicator enzyme
catalyzes the final reaction
indicator enzyme
method for couple ENZ assay
measures single absorbance at fixed time, reaction is assumed to be linear over fixed time
fixed time
method for couple ENZ assay
multiple absorbances are measured
Continuous monitoring/kinetic assay
method for couple ENZ assay
more accurate because any deviation from linearity is readily observed
Continuous monitoring/kinetic assay
*most common cause of deviation from linearity
occurs when the enzyme is so elevated that all substrate is used early in the reaction time
Units to report enzyme levels when enzymes are quantified relative to
their activity rather than a direct measurement of concentration
Activity Units
amount of enzyme that will catalyze the reaction of 1umol of substance per minute under specified conditions
IU
Enzyme activity SI unit –
katal (mol/s)
How many nkat is in 1.0IU?
1.0 IU = 17nkat
When will enz act as REAGENTS
measure non enzymatic constituents like glucose, cholesterol
Enzymes as __
Used for methods quantifying substrates for the corresponding enzyme
Reagent
– chemically bonded to adsorbents such as agarose
Immobilized Enzyme
Systematic name of CK
Creatine-N-phosphotransferase
MW of CK
82000 Daltons
ENZ assoc with ATP regeneration
CK
Creatine + ATP (catalyzed by CK) will yield
creatine phosphate + ADP
minor tissue sources of CK
bladder, placenta, GIT, thyroid, uterus, kidney, lung, prostate, spleen, liver, pancreas
CK levels are elevated in (3)
MI
Rhabdomyolysis
Muscular dystrophy
CK isoenz
– brain type, migrates fastest to the
anode
CK-BB
CK isoenz
Cannot pass blood-brain barrier, undetectable in blood
CK-BB
CK isoenz
Elevated in patients with various carcinomas; may be useful tumor associated marker
CK -BB
– hybrid type, less than 6% of total CK
CK-MB
CK-MB – hybrid type, less than ?% of total CK
6
are more sensitive markers of MI; they are released earlier and persist longer
Troponin I and T
CK Isoenz
Following myocardial infarction, levels rise within 4-8 hours, peak at 12-24 hours, and return to normal after 48-72 hours
CK-MB
CK isoenz
– muscle type, most common, migrates slowest to the anode
CK-MM
atypical ck
– migrates midway between CK-
MM and CK-MB
Macro-CK
atypical ck
CK-MM complexed with IgG, or CK-MM complexed with lipoproteins
Macro-CK
atypical ck
In serum, 0.8-1.6%
Macro-CK
atypical ck
Age and sex related
Macro-CK
atypical ck
– bound to exterior surface of the inner mitochondrial membranes of muscle, brain, and liver cells
Mitochondrial CK (CK-MI)
enzyme released from hemolyzed RBCs and appearing as a band cathodal to CK-MM
adenylate kinase
may cause falsely elevated CK or CK-MB value
adenylate kinase
– more sensitive and precise than electrophoresis if done properly
Ion-exchange chromatography
ref mtd of isoenz meas
EF
– measure enzyme concentration rather than enzymatic activity,
Immunoassays (Radioimmunoassay, immunoinhibition)
mtd of isoenz meas
can detect inactive forms of enzymes
Immunoassays (Radioimmunoassay, immunoinhibition)
CK MEAS
Tanzer Gilvarg coupled with
PK-LD-NADH system
CK MEAS
Tanzer Gilvarg pH
9.0
CK meas
Oliver Rosalki coupled with
HK-G-6-NADP system
CK meas
Oliver Rosalki pH
6.8
Tanzer gilvarg
ADP + ____◄PK► pyruvate + ATP
phosphoenolpyruvate
Tanzer gilvarg
ADP + phosphoenolpyruvate ◄PK► __ + ATP
pyruvate
Tanzer gilvarg
Pyruvate + NADPH + H+ ◄LD► ____ + NAD+
Lactate
CK meas
2 to 6 times faster than forward reaction
Oliver Rosalki
Oliver Rosalki
Creatine phosphate + __◄CK► creatine
+___
Creatine phosphate + ADP ◄CK► creatine
+ATP
Oliver Rosalki
ATP + __ ◄HK► ADP + ___
ATP + glucose ◄HK► ADP + G-6-P
Oliver Rosalki
__ + NADH ◄G-6-PD►__ + NADPH
G-6-P + NADH ◄G-6-PD► 6- phosphogluconate + NADPH
CK errors
___ mg/dL hemoglobin interferes
> 320
Errors in CK meas
Instability of CK – rapid inactivation of enzyme due to oxidation of ___ group
sulfhydryl
Errors in CK meas
Instability of CK
Can be partially reversed by adding
sulfhydryl compounds: N- acetylcysteine, mercaptoethanol, thioglycerol, dithiotreitol
– inactivates CK
Light exposure
Light exposure inactivates CK Activity can be restored if specimen is (2)
1) stored in the dark at 4°C/7 days
2) 20°C/month if the assay used a sulfhydryl activator
higher muscle mass results in __ CK levels
higher
RR of ck M
15-160 U/L
RR of ck F
15-130 U/L
