Enzyme assays The amount of enzyme protein present can be determined in terms of the catalytic effect it produces, that is the conversion of substrate to product 1. The overall equation of the reaction 2. The disappearance of substrate or the appearance of product 3. Cofactors, pH temperature 4. Sufficient supply of substrate
Enzyme assays • The amount of enzyme protein present can be determined in terms of the catalytic effect it produces,that is the conversion of substrate to product. 1. The overall equation of the reaction 2. The disappearance of substrate or the appearance of product 3. Cofactors,pH ,temperature 4. Sufficient supply of substrate
Enzyme assay Enzyme activity(酶催化某一化学反应的能 d): the rate of appearance of product or the rate of disappearance of substrate Absorbance: spectrophotometer分光光度计 Two most common molecules. NADH(reduced nicotinamide adenine dinucleotied还原型烟酰胺腺嘌呤二核苷酸) NADPH: 340nm
Enzyme assay • Enzyme activity（酶催化某一化学反应的能 力）:the rate of appearance of product or the rate of disappearance of substrate • Absorbance:spectrophotometer分光光度计 • Two most common molecules: • NADH(reduced nicotinamide ademine dinucleotied还原型烟酰胺腺嘌呤二核苷酸) • NADPH:340nm
NADH(NADPH) site of hydrogen atom addition N positive charge OH OH denine OCH D nucleotide OH OH OH OH
NADH (NADPH)
LDH: Lactate dehydrogenase H-C-H Adenine H Pyruvate NADH NH N HO-C-H LDH H-C-H Adenine Lactate NAD
LDH:Lactate dehydrogenase
Linked enzyme assays If neither the substrates nor products of an enzyme-catalyzed reaction absorb light at an appropriate wavelength, the enzyme can be assayed by linking to another enzyme-catalyzed reaction that does involve a change in absorbance The second enzyme must be in excess, so that the rate-limit step in the linked assay is the action of the first enzyme
Linked enzyme assays • If neither the substrates nor products of an enzyme-catalyzed reaction absorb light at an appropriate wavelength,the enzyme can be assayed by linking to another enzyme-catalyzed reaction that does involve a change in absorbance. • The second enzyme must be in excess,so that the rate-limit step in the linked assay is the action of the first enzyme