Enzymes

By following the concentration of substrate or product change with time Following the progress of the reaction the substrate will decrease and the product will build up

Artificial substrates

As enzymes work so fast we use artificial substrates This slows the reaction down so we can easily measure the rate

As enzymes work so fast we use artificial substrates This slows the reaction down so we can easily measure the rate

The reaction may not start immediately when the reagents are mixed There may be a lag time required to permit formation of a measurable product Typically, the most accurate value is obtained when the reaction just gets going i.e. initial velocity

We are measuring the change of absorbance over time or the rate at which the absorbance changes It is critical to measure the activity of the enzyme when the reaction rate depends only on the amount of enzyme present (first order of kinetics) This is the linear phase of the progress curve

It is critical to measure the activity of the enzyme when the reaction rate depends only on the amount of enzyme present

Reaction progress per unit time

A = rate is constant B = rapidly becomes non-linear C = there is a bigger lag phase

Reactions linked together such that a second reaction generates a compound which can be measured spectrophotometrically The indicator reaction needs to be directly proportional to the rate of reaction (product formation) in the primary reaction

Measurement of AST

Primary assay reaction Indicator reaction

Urease Uricase Glucose Oxidase Perioxidase Hexokinase Cholesterol Oxidase Lipase Alkaline phosphatase Reverse transcriptase

Can also exploit the specificity of an enzyme to measure the concentration of a substance Typically the enzymes substrate e.g. a common method to measure glucose based on glucose oxidase - peroxidase (GOD-PAP)

An end point method/equilibrium method Using a coupled system to produce a colour The glucose oxidase gives the specificity The peroxidase generates the colour A trapping reaction is necessary to ensure that the enzyme will quantitatively convert substrate to product This traps the product and makes the reaction go to completion

The production of H2O2 by an oxidase is followed by production of the red compound quinoneimine (cholesterol, TAGs, glucose) The consumption of NADH is followed by a decrease in absorbanace at 340nm (ALT, AST) Release of the yellow coloured nitroanilide (GGT, ALP)

Specificity -> contrast with chemical methods Sensitivity -> nmol and pmol amounts easy to do Versatility -> using coupled systems we can assay virtually anything Reproductibility -> as long as the same amount of enzyme is added to the tube the reaction rate will be the same Ease of use -> enzymes like mild conditions: most chemical methods need harsh ones

Enzymes can also be immobilised onto surfaces Allow for the measurement of the enzymes substrate (specific analytes)

Immobilised enzyme: used for the detection of abnormal substances in urine e.g. Paper coated with GOD-PAP for glucose in urine

Enzyme concentration Substrate concentration pH Temperature Inhibitors and activators Coenzymes and prosthetic groups Timing

One analyte may have a large number of different methods or different assay conditions used to measure it This makes it difficult to compare results from different labs This also makes it more difficult for clinicians to interpret results

Comparable results Common units Common reference ranges Indication of trueness (accuracy)

Enzyme preparation is generated with known catalytic activity Used as a standard (reduces variability) Leaves a path of ‘traceability’ patient results can be traced back to the original standard Requires co-operation between international bodies, manufacturers, labs

By optimising: -pH - Temperature - Substrate conditions - Method parameters (kinetic versus endpoint) - Units