At Citeq Biologics, we are now performing several protease assays, next to the major allergen determinations, to get a more comprehensive characterization of our allergen extracts.
It is very important to define extracts of allergen sources. Most researchers in the field of allergy have mostly been focusing on the amount of total protein and on the presence of major allergens. Of course, these are key values. Whilst focusing on these, we tend to forget about other potential factors with relevance to allergy, like proteases. It is known that many allergens have enzymatic activity. Protease activity is the most common and most described one. Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants by stimulating protease-activated receptors on epithelial cells. Enzymatic activity, therefore is an important parameter to characterize the allergenicity of an extract. Besides that, it also affects stability of allergen extracts.
Proteases (also called proteinase or peptidases) are enzymes that catalyze the breakdown of proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. (López-Otín & Bond, 2008) Proteases break the peptide bonds between amino acids by adding hydrolysis; a water molecule. Hydrolysis of proteins is called proteolysis. There is a difference between endoproteases and exoproteases. An endoprotease can cut in the middle of a protein chain, whilst an exoprotease can only remove an amino acid at the end of a chain.
The nucleus of a protease is a chemically active group. This attacking group is part of an amino acid such as cysteine, serine or aspartate. The other amino acids in the active center help to catalyze the hydrolysis reaction. There are many different families of proteases. Proteases can be classified into seven broad groups:
• Serine proteases – using a serine alcohol
• Cysteine proteases – using a cysteine thiol
• Threonine proteases – using a threonine secondary alcohol
• Aspartic proteases – using an aspartate carboxylic acid
• Glutamic proteases – using a glutamate carboxylic acid
• Metalloproteases – using a metal, usually zinc
• Asparagine peptide lyases – using an asparagine to perform an elimination reaction (not requiring water) (Oda, 2012)