Alpha-1 Proteinase Inhibitor (A1PI) Activity Assay

Introduction

Alpha-1 proteinase inhibitor (A1PI), also known as alpha-1 antitrypsin (A1AT) and alpha-1 antiproteinase (A1AP) is the archetypal member of superfamily serine protease inhibitors (SERPINs). A1PI is the only known inhibitor of elastase but also inhibits trypsin and chymotrypsin. As an acute phase protein, A1PI is commonly associated with regulation of inflammatory reactions, especially in lung tissue where its main function is to inhibit neutrophil elastase. Binding of the target protease to the reactive center loop (RCL) of A1PI is irreversible and proteolysis results in a conformational change rendering A1PI inactive.

Another notable feature of A1PI is that its activity can be regulated by oxidation of the sulfur containing amino acid methionine. Reactive oxygen and nitrogen species (ROS & NOS) can both react with A1P to create methionine sulfoxide (MetO) residues at key sites resulting in inactivation of the enzyme. Regulation of A1PI in this fashion is reversible by the action of methionine sulfoxide reductase, an important regulatory enzyme of interest in redox related research. The action of ROS and NOS in helping to regulate the activity of cellular housekeeping proteins such as A1PI is fast becoming a major area of interest in researching many disease states.

In contrast to ELISA measures of total protein, monitoring of A1PI activity levels can provide useful information about the effect of ROS and NOS on actual enzyme function. Measurement of A1PI activity in both control and research samples can be useful as an index of oxidative stress effect on proteins.

Test Principle

Alpha-1 proteinase inhibitor rapidly, specifically and irreversibly binds to the active site of elastase. The molar reaction ratio is 1:1 such that measuring inhibition of elastase activity becomes a convenient method for quantifying A1PI activity. In Phase One of this assay a known quantity of elastase is allowed to react with sample A1PI. Elastase Reagent is provided in excess such that the remaining elastase activity can be measured as a means of determining the level of A1PI mediated inhibition.

Elastase]Known+ [A1PI]Sample = [Elastase/A1P1]Complex + [Elastase]Test

In Phase 2 of the assay N-succinyl-(Ala)3-p-nitroanilide (NSAN) reacts with the remaining Elastase to cleave p-nitroanaline with a maximal absorbance at 410 nm. Remaining sample elastase is determined by comparing sample A410 to a five point standard curve generated at the time of sample testing. The amount of elastase inhibition is found by subtracting the remaining test sample elastase (measured) from the known elastase quantity (as shown on the vial).

[Ei] = [Ek] - [Et]

Since A1PI and elastase react in a one to one molar ratio, the concentration of A1PI in the reaction mix is the same as the calculated elastase inhibition.

[A1PIRxm] = [Ei]

A1PI concentration in the original sample is therefore calculated as:

[A1PIs] = [Ei] * Sample Dilution