analytical Applications of enzymes

Chem*4570 Applied Biochemistry Lecture 25 Analytical Applications of enzymes Clinical laboratory: many tests are based on measuring levels of enzymes e.g. SGOT serum glutamate-oxaloacetate transaminase. The enzyme is normally abundant in cardiac muscle or liver tissue. Presence in serum is an indicator of tissue damage: 10- to 100-fold increase after heart attack, viral hepatitis or toxic necrosis of liver. The level is monitored to indicate severity of damage. However none of the substrates are particularly easy to measure directly. The assay is done by linking the reaction to a second indicator reaction, in which NADH is consumed. transaminase aspartate + α-ketoglutarate ‡ oxaloacetate + glutamate malate dehydrogenase oxaloacetate + NADH + H+ ‡ malate + NAD+

primary reaction indicator reaction

The enzyme for the primary reaction is present in the clinical sample. This is added to its substrate mixture plus the indicator enzyme and NADH. As the primary reaction proceeds, oxaloacetate is produced and is rapidly reduced by NADH in the indicator reaction. The decrease in NADH is detected by change in absorbance at 340 nm; NAD+ does not absorb above 300 nm. For this to work, the indicator enzyme should be present at 10 to 100 times the maximum expected activity of the transaminase to ensure that there is no lag in oxaloacetate reduction, and the primary reaction is rate limiting and is the only factor determining the measured rate. It is also important that the indicator reaction has a large equilibrium constant so that there is virtually no reverse reaction, and that a minimal concentration of oxaloacetate persists. For malate dehydrogenase, ∆Go ’ = –29.7 kJ/mol, so Keq = 1.6 × 105 . Other common clinical enzyme tests: glutamate-pyruvate transaminase indicates liver damage by hepatitis or necrosis alkaline phosphatase liver isozyme indicates cholestasis or bile duct blockage gamma glutaminyl transpeptidase indicator of cholestasis creatine kinase heart isozyme indicates possible myocardial infarct serum a-amylase acute pancreatitis acid phosphatase prostate carcinoma Other enzymes may be used to detect levels of specific analytes: glucose by glucose oxidase cholesterol by cholesterol oxidase urea by urease.

Measurement of specific hexoses can also be important to monitor progress of reactions in industrial processes. Accurate enzyme assay by classical methods requires a certain degree of skill, and skill represents high-priced labour. One objective in analytical techniques has been to simplify assay procedures so that they can be performed by less skilled workers, or in the case of simple clinical tests like blood glucose, by the patients in their own home. Enzyme electrodes Measuring pH is a simple procedure with appropriate instrumentation, using electrodes which have been designed to respond only to the presence of hydrogen ion. Enzyme electrodes provide the same simplicity for certain analytes and potentially can be reduced to microchip form for automation. Example: monitor protease digestion at pH 9 (suitable for trypsin-like proteases). At pH 9, the N-terminal of a polypeptide chain (pKa = 8) is deprotonated and neutral, whereas the C-terminal is deprotonated and negative. Each time a polypeptide is cut, a new C-terminal is exposed and one H+ is released. This can be monitored by pH shift: simple but needs calibration to determine buffering capacity ∆pH/∆[H+] Autotitration has also been used: NaOH is added in precisely measured amounts to maintain pH constant at 9.000 This system can be adapted not to measure the level of substrate present in a sample rather than the activity of the enzyme. Enzyme is immobilized on a gel disk placed in contact with the pH sensitive surface of a glass H+ electrode. The enzyme disk is held in place by a porous membrane freely permeable to the substrate. Reaction occurs as substrate diffuses into the enzyme disk. A disadvantage is slow approach to steady reading and slow response due to the need for substrate to diffuse into the enzyme matrix, although ultrathin or granular matrices have improved performance.

Basis of ion-sensitive potentiometric electrodes Consider a chamber separated into two compartments by a membrane. One compartment contains 1.0 M HCl and the other 0.01 M HCl. 1) The membrane is totally impermeable. The two sides maintain their concentration Nothing passes to the other side Potential across membrane = 0 V 2) The membrane is permeable to H+ and Cl– The more concentrated solution diffuses to the right hand side due to the osmotic difference. Membrane potential = 0 V 3) The membrane is selectively permeable to H+ but not Cl– . H+ starts to permeate the membrane due to concentration difference, but Cl– is left behind. A membrane potential rapidly develops to prevent imbalance of positive and negative ions in the two solutions. The equilibrium membrane potential Ψ just balances the osmotic tendency of H+ to move towards the lower concentration, and will be positive on the low concentration side. Ψ = -2.3 RT/F log10 [H+]L/[H+]R = 2.3 ∆ pH RT / F = 0.059 ∆ pH Thin glass makes an effective selectively permeable membrane for H+ ions. H+ moves by “hopping” from silicate to silicate, and does not need a clear channel through the glass. No other ion can do this at any effective rate. Membranes selective for other ions can be made from polymer containing ionophore inclusions. Ionophores are organic molecules designed to replace the hydration shell around an ion, and are selective based on ionic radius and charge density. This provides a basis for assays where the ion released is other than the H+ ion, e.g. urease NH2 -CO-NH2 + 3 H2 O → 2 NH4 + + HCO3 – + OH–