Enzymology - Lab Flashcards
activities in the body that involves enzyme
- glycolysis
- creation of heme in hemoglobin
- coagulation cascade
- beta oxidation
- gluconeogenesis
proteins within cells
enzymes
why enzyme is used as catalyst?
because they make the chemical reaction faster
does glycolysis still happen even without enzyme?
yes, but it takes 300,000 years for the action to be completed
general properties of enzyme
active site: water free
allosteric site: cavity other than the active site
a molecule or a substance where an enzyme acts
substrate
factors of enzymology
- enzyme concentration
- substrate concentration
- cofactors
- inhibitors: competitive, non-competitive, uncompetitive
- isoenzymes
- temperature
- storage
non-protein that must bond to a particular enzyme before a reaction occurs
cofactors
electrolytes that are commonly used as cofactor in enzymatic reactions
- magnesium
- chloride
- calcium
temp where enzymes are activated
25 - 37C
temp where enzymes are being denatured
38-50C
temp where enzyme will be inactivated
60-65C
does not compete bcos the inhibitor will bind to other site
non-competitive
binds w/ the enzyme substrate complex (no product/reaction will happen)
uncompetitive
enzymes w/ similar enzymatic activities but differ their structure and origin
isoenzyme
what is a fixed time
reacts at a designate time
multiple measurements of absorbance changes are made during the reaction
kinetic assay
enumerate the cardiac enzymes
- Creatine-Kinase ATP-Creatine-N-Phosphotransferase
- Lactate dehydrogenase L-Lactate: NAD Oxireductase
CK is an enzyme important in the?
regeneration of adenosine triphosphate
why CK is most commonly used in the diagnosis of AMI, muscular dystrophy, and CNS disorders?
bcos they are found in skeletal muscles, heart muscles, and brain tissues
CK is dimeric because?
has 2 different monomers, the “M” and “B”
transfers phosphate group between creatine phosphate and ADP
creatine kinase
creatine kinase is also known as?
ATP-Creatine-N-Phosphotransferase
CK is most commonly used in the diagnosis of?
- AMI
- muscular dystrophy
- CNS disorders
normal values for CK in males and female
male: 25-90 IU/mL (0.42-1.51 mmol/L)
female: 10-70 IU/mL (0.17-0.18 mmol/L)
sample used for testing CK
serum
anticoagulants inhibit CK
isoenzymes of creatine kinase “ATP-Creatine-N-Phosphotransferase” (from fastest)
- CK BB “brain type” CK1
- CK MB “hybrid type” “CK2”
- Macro CK
- CK MM “muscle type” “CK3”
- CK-Mi “mitochondrial CK”
isoenzyme abundant in cardiac and skeletal muscle (major activity is in the heart)
CK-MM “muscle type” “CK3”
major isoenzyme in sera of healthy people
CK-MM “muscle type” “CK3”
CK-BB is __ type “CK-3” which is normally found in?
neonatal sera rare in adult serum
CK-BB elevates in
brain injury and carcinomas
this isoenzyme is with significant amount in the heart and most specific for myocardial damage
CK-MB CK-2 “hybrid type”
CK-MB “hybrid type” CK-2
elevates:
peaks at:
normalize in:
elevates: 4-8 hrs
peaks at: 12-24 hrs
normalize in: 48-72 hrs
this isoenzyme is falsely elevated in hemolysis
CK-MB CK-2 “hybrid type”
Macro-CK is common in ___ migrates middle of ___ and ___
Macro-CK is common in older woman migrates middle of ** middle of MM and MB** ___.
CK-Mi is also known as
mitochonrial CK
migrates cathodal to CK-MM
mictochondrial CK (CK-Mi)
mitochondrial CK migrates cathodal to CK-MM which indicates ____
several illness
enumerate the methods for CK
- Tanzer and Gilvarg Assay - forward
- Oliver Rosalki - reverse
- Electrophoresis
forward method of CK
ph:
absorbance:
Tanzer and Gilvarg Assay
ph: 9
absorbance: 340nm
reverse method of CK
ph:
absorbance:
CK Method
Oliver Rosalki - reverse
ph: 6.8
absorbance: 340 nm
what is electrophoresis?
separation of charged compounds
(ex: serum protein, hgb, isoenzyme)
cathode
movement towards negative region
anode
movement towards positive region
Lactate dehydrogenase is also known as?
L-Lactate: NAD oxidoreductase
a transferase that hastens the interconversion of ____
Lactate dehydrogenase
a transferase that hastens the interconversion of lactic acid and pyruvic acid
LDH is found in?
various tissue sources
LDH is composed of ____ of two types: “M” (A) and “H” (B)
4 peptide chains
highest levels of LDH are detected in what diseases?
pernicious anemia and hemolytic disorders
disease associated with elevations of LDH are?
- hepatic disorders
- AMI
- pulmonary infarct
- acute lymphoblastic leukemia
forming glucose from non-carbohydrate sources
gluconeogenesis
isoenzymes
- LDH1
- LDH 2
- LDH 3
- LDH 4
- LDH 5
- LDH 6
- LDH1 (HHHH)
- LDH 2 (HHHM)
- LDH 3 (HHMM)
- LDH 4 (HMMM)
- LDH 5 (MMMM)
- LDH 6
major isoenzyme in healthy people
most abundant and heat stable
LDH 2 (HHHM)
an arteriosclerotic cardiovascular failure marker
LDH6
these LDH can be found ___
- LDH1 =
- LDH 3 =
- LDH 4 =
- LDH 5 =
- LDH1 = heart, rbc, kidneys
- LDH 3 = lungs, pancreas, spleen, lymphocytes
- LDH 4 = skeletal muscle, liver, intestine
- LDH 5 = liver, skeletal muscle, intestine
LDH can be used as a cardiac marker for these diseases, enumerate
- myocardial infarction
- hepatitis
- hemolysis
- lung and muscle disorders
LDH in myocardial infarction
elevates:
peaks:
remains:
elevates: 12 - 24 hrs after onset
peaks: 48 - 72 hrs
remains: 10 days
for emergency diagnostic, what cardiac enzyme u will check?
Creatine kinase since it is the one who elevates faster
for delayed diagnosis, what enzyme should be check
lactate dehydrogenase
enumerate the LDH method
- forward or direct:
- reverse or indirect:
- forward or direct: Wacker method
- reverse or indirect: Wroblewski method
a direct LDH method
pH:
absorbance:
Wacker Method
pH: 8.8
absorbance: 340 nm
LDH Method
Wroblewski Ladue
pH:
absorbance:
pH: 7.2
absorbance: 340 nm
3x faster than wacker
indirect/reverse method of LDH
what are the other cardiac markers?
- myoglobin
- troponin I
- brain-type natriuretic peptide
transport and stores oxygen to intracellular respiratory enzymes of contractile cells
myoglobin
other cardiac marker with high sensitivity
myoglobin
myoglobin markers of AMI
- elevates ___ after onset
- peaks at _____ hours
- normalizes ___
- elevates 2-3 after onset
- peaks at 8-12 hrs hours
- normalizes 18-30 hrs
troponin I is found in ___ and with greater cardiac specificity
myocardium
troponin markers of AMI
- elevates ___ after onset
- peaks at _____ hours
- normalizes ___
- elevates 3-6 hrs after onset
- peaks at 12-18 hours
- normalizes 6 days
what is brain-type natriuretic peptide?
not and enzyme but heart failure marker
amylase is also known as?
Alpha 1-4 Glucan 4 Glucohydrolase
considered as the smallest enzyme
Alpha 1-4 Glucan 4 Glucohydrolase
amylase
AMYLASE
activators:
substrate:
activators: calcium and chloride
substrate: starch or glycogen
acinar cells of the pancreas releases ___
amylopsin
salivary glands releases __
ptyalin
amylase clinical significance
- acute pancreatitis
- intestinal obstruction
- cholecystitis
- acute appendicitis
AMYLASE
elevates:
peaks:
normalizes:
elevates: 2-12 hrs after onset
peaks: 24 hrs
normalizes: 3-5 days
measures reducing sugar from starch breakdown?
amylase method
Saccharogenic
- Measure ____ activity by following decrease in substrate concentration
- Follows breakdown of starch
Amyloclastic
Indicator of amyloclastic
Iodide
uses dye to check amylase activity
Chromogenic
Lipase is also known as
Triacylglycerol acylhydrolase
Lipase needs the presence of?
Bile salts and colipase
- Hydrolyze glycerol esters of long-chain fatty acids
- Most lipase activity found in serum are usually from the pancreas and some
are secreted by gastric and intestinal mucosa
Lipase
Lipase clinical significance
- Acute Pancreatitis
- Perforated or duodenal ulcer
- Intestinal obstruction
- mesenteric vascular obstruction
Cherry Crandal
reference method for Lipase
Cherry crandal utilizes __ for hydrolysis
Indicator:
Use olive oil for hydrolysis
Indicator: Phenolphthalein
substrate: olive oil, triolein (result: fatty acid PINK)
Most lipase activity found in serum are usually from the ___ and some are secreted by ___.
Most lipase activity found in serum are usually from the pancreas and some
are secreted by gastric and intestinal mucosa
