Re: How does lactoperoxidase label Tyrosine residues?

hi Angela,

Ah, this is classic biochemistry - investigation of membrane protein topology in erythrocytes using radiolabelling.

The mechanism of this reaction is not too complex, although there are many papers in the literature that discuss it. Lactoperoxidase is a heme-containing enzyme which, like other peroxidases, catalyses the reduction of hydrogen peroxide to water. This is a redox reaction - if something gets reduced then something else gets oxidised. If we add iodide ions to the mix, they become oxidised to iodine:

2I^- + H₂O₂ + 2H⁺ --> I₂ + 2H₂O

Lactoperoxidase also catalyses the oxidation of phenols and aromatic amines. In this reaction, the iodide binds to the enzyme and forms a highly reactive species which then iodinates free tyrosine or tyrosyl residues in peptides.

It's not clear from your question whether you expected your glycophorin to be labelled (i.e. if other people have done this). If so, my first suggestion would be to check all of the reagents - is the enzyme active? Is the activity of the radioiodine high enough? Are all the buffers the correct pH/ionic strength? Are the reagents in the correct proportions?

Some proteins are certainly more amenable to radioiodination than others. This depends largely on the solvent accessibility of the tyrosyl - how exposed it is.

I found a heap of references to this technique using Google and PubMed searches. Try "radioiodination lactoperoxidase tyrosine" for starters, then add some search term qualifiers like "mechanism". I've included a few of the most useful references at the end of this reply. Good luck and remember, don't despair when those experiments don't work out first time!

References
Lactoperoxidase information from Worthington Biochemical - with lot of references
An enzymic method for the trace iodination of immunoglobulins and other proteins - the original study
iHOP information for lactoperoxidase - lots of references
Studies on the mechanism of the iodination of tyrosine by lactoperoxidase
Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin - notes about accessibility of Tyr residues
The organization of the major protein of the human erythrocyte membrane
Optimal conditions for lactoperoxidase catalyzed radioiodination of external proteins on mouse erythrocytes