Lab Quiz 3 - Measurement of Multiple Reaction Types

absorbance pattern of biomarkers

What is measured in clinical chemistry

Endpoint Colorimetric

Endpoint Enzymatic

Kinetic Reactions

3 types of Chemical Reactions:

Endpoint Colorimetric Spectrophotometry

measures a colored product that is formed by a chemical reaction with the chemical of interest

measures visible light

ex: Glucose, Cholesterol, Triglycerides, Creatinine

complementary color

What color should we set our spectrophotometry for max absorbance

**hint: if we measure blue, we set to orange

Endpoint Enzymatic Spectrophotometry

measures an enzyme's activity

can measure a chemical compound as a result of enzymatic cleavage, or coenzymes (produced or consumed)

ex: Alkaline phosphatase, Glucose hexokinase

Kinetic Spectrophotometry

takes multiple measurements as the reaction is going

measured as average absorbance change per minute

Significance of Kinetic Measurements

measurement of enzymes in body fluids is most clinically significant when the results closely relate to recent release from diseased or damaged tissue

more measurement = more accurate

ex of clinically sig enzymes: AST, ALT, ALP, CK, GGT

organ or tissue damage before pt exhibits outward signs of disease

Serum enzyme activity is often a sensitive predictor of:

the ability of an active enzyme to catalyze/accelerate the rate of a chemical reaction

methods of enzyme analysis involve measuring:

change in absorbance per min

In kinetic spectrophotometry, the rate of product formation is determined by:

first order

rate is directly proportional to concentration of substrate

doubling the concentration increases the rate by a factor of 2

concentration of substance and availability of enzyme

First order is dependent on what 2 things:

zero order

rate is independent of the concentration of substrate

doubling the concentration has NO effect on rate

aka max velocity

only only dependent on the amount of enzyme available

Zero order is dependent on:

change in absorbance remains constant

Kinetic analysis is most accurate when:

zero order

When is measurement made in kinetic analysis?

all active sites on the enzyme are filled with substrate

Zero-order kinetics is the most accurate indication of enzyme activity because:

quaternary structure

Structural differences include differences in peptide structure of one of several protein chains of which structure of a protein unit

Isoenzyme

forms of the same enzyme that arise from unique gene sequences

specificity

Isoenzymes increase the ___________________ of a method

Electrophoresis

Most common lab testing for isoenzymes

other: chromatography and immunochemistry methods