Tabares, MA©

ENZYME ASSAYS Procedure/description wavelength

CK Oliver-Rosalki-Hess Inc in absorbance of NADPH produced by the G6PD reaction 340nm

Tanzer and Gilvarg Dec in absorbance due to consumption of NADH 340nm

*Disadv: the initial 340nm abs in the system is high

LD Wroblewski-Cabaud (colorimetric) 440-525nm

The pyruvate produced is allowed to react with DNPH to

produce a golden brown phenyl hydrazine solution at an
alkaline pH.
Wroblewski-LaDue Conversion of pyruvate to lactate. 340nm
(spectrophotometric) NADH acts as a co-substrate, the decrease in absorbance is
monitored.
NV of pyruvate to lactate: 80-280 U/L at 37C
Wacker (spectrophotometric) Conversion of lactate to pyruvate.
NADH is generated during the reaction, the increase in
absorbance is monitored.
NV: 100-225 U/L at 37C
AST and Frankel-Reitman (reaction w/ Ketoacid formed after enzymatic reaction is allowed to react w/ 505nm
ALT DNPH) DNPH to form a blue derivative
Babson (coupling w/ diazonium salt) Violet diazonium derivative is formed
*subject to a lot of interference
Karmen or walker (coupled enzyme Keto acid formed is allowed to react w/a system using NADH.
reaction) Reduction in abs measure the activity of the enzyme in the
sample.
*preferred method

ALT: pyruvate formed is converted to lactate by the LD

enzyme.
AST: oxaloacetate produced is reduced to malate by the enzyme
malate dehydrogenase.
*both involve consumption of NADH
ALP Bodansky (Kay and Bodansky) Substrate:B-glycerophosphate
End products: Inorganic phosphate
Comments: Long incubation time
Shinowara-Jones-Reinhart Substrate: B-glycerophosphate
End products: organic phosphate
Comments:high blank values; long incubation time
King-Armstrong Substrate: Phenylphosphate
End products: Phenol
Comments: endpoint; requires protein removal
Bessey-Lowry-Brock Substrate: p-nitrophenyl phosphate
End products: p-nitrophenol or yellow nitrophenoxide
Comments: endpoint or kinetic; rapid
Bowers:McComb Substrate: p-nitrophenyl phosphate
End products: p-nitrophenol or yellow nitrophenoxide
Comments: uses phosphate-accepting buffer
Klein-Babson-Reed Substrate: buffered phenolphthalein phsophate
End products: free phenolphthalein
Higgins and Talalay Substrate: Phenolphthalein diphosphate
End products: phenolphthalein
Moss Substrate:α-naphthyl phosphate
End products: α-naphthol
ACP Bodansky Substrate: B-glycerophosphate
Product: Inorganic Phosphate & glycerol
Complements: Lenghthy assay & non specific

Gutman &Gutman
King-Armstrong
Hudson
Babson & Reed
Bessey-lowrey-Brock
Roy
Reitz- Guilbault
LAP Goldbarg-Rutenburg method
GGT Szasz method
Amylase Amyloclastic Method (aka
Iodimetric method)
Caraway method
Saccharogenic method
Amylometric method
Chromometric method
Chromolytic method
Coupled kinetic method
Lipase Tirimetric: Cherry-Crandall
method
Henry-Sobel-Berkman method
(modified Cherry-Crandall)
Turbidimetric: Shihabi-Bishop and
Sigma-Teitz methods
CHS pH change: Michel method
Colorimetric: Ellman method
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Substrate: Phenylphosphate
Product: Phenol
Complements: Non-specific
Substrate: PNPP
Product: P-nitrophenol (yellow)
Complements: Non-specific; rapid
Substrate: α-naphthol
α-naphthol
Complements: Complicated; less sensitive
Substrate: PNPP
Product: P-nitrophenol(yellow)
Substrate: Thymolphtahlein monophosphate
Product: Thymolphtahlein (blue)
Complements: More specific for prostatic form
Substrate: 4-methyl umbilliferone phosphate
Product: 4-methyl umbilliferone
Complements: Fluorescent; some improved sensitivity
β naphthylamide produced by enzymatic activity is allowed to
react with ethylene diamine dihydrochloride to form a blue
product
Uses p-nitroanilide s the substrate.
This measures the decrease in substrate concentration.
Indicator: I2
The extent of hydrolysis of starch substrate the enzyme
amylase is measured by the loss of the blue color when iodine is
mixed with starch.
Measures the products based on their reducing properties.
measures the amount of starch hydrolyzed within a specified
time.
measures the time required for complete hydrolysis of a
substrate.
measures the amount of soluble dye released from an insoluble
starch-dye complexed.
example of this is the Teitz method.
Lipase hydrolyzes TAGs (tri-olein) in olive oil. After overnight
incubation the fatty acid released is titrated w/ NaOH using
phenolphthalein as indicator
pH meter or a thymolphthalein is used. Incubation period is
16hrs.
Uses olive oil as substrate. Decrease in turbidity is measured.
Temperature sensitive. When choline ester is hydrolyzed, a
proton in released. Monitored by pH meter or electrometric
titration.
Cholinesterase is allowed to react w/ a thiol ester to for a thio 410nm
product instead of an alcohol. Thiolcholine produced reacts w/a
disulfide called dithiobis-nitrobenzoic acid(DNBA) which is a

G6PD Ascorbate cyanide test
Fluorescent spot test
Colorimetric test: Agar-formazan
method
Colorimetric test: Vial-formazan
method
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colored product.
*rapid and sensitive and a recommended method
Blood incubated w/a soln of sodium cyanide and
sodiumascorbate, hydrogen peroxide is generated from the
coupled oxidation of ascorbate and Hb. Cyanide inhibits
catalase,hydrogen peroxide is available to oxidize Hb, and the
brown color of metHb is discernable.
*occur more rapidly in G6PD deficient cells
Filter paper is spotted with mixture and is exposed to a UV
lamp to observe for fluorescence
*30-60mins
*NADPH – fluorescence; NADP – no fluorescence
*G6PD(+) – NADPH; G6PD(-) - NADP
*recommended by ICHS
*not specific: (+) PK deficiency and unstable Hb
*most sensitive
Rgts are mixed w/ the agar and allowed to solidify. A special
filter paper disk containing the sample is placed in the agar.
Results after 24hrs incubation.
*presence of G6PD: violet ring
Deficient: yellow ring
Rgts in soln are placed in a microcentrifuge tube. Fresh
capillary or WB sample or sample collected in a special filter
paper (e.g., Guthrie filter paper) is added into the soln. invert
vial several times and observe color change w/in 5-10mins.
*presence of G6PD: violet color
deficient: yellow soln