An Unique Aggregated Protein
Keyhole Limpet Hemocyanin (KLH) is one of the most commonly used carriers in the conjugation of peptides for
antibody production. It is a copper-containing protein that belongs to a group
of non-heme proteins called hemocyanins, which are found in arthropods and
mollusca. KLH is isolated from the mollusc Megathura crenulata.
Molecular weight
Molecular weight: MW of
KLH is 4.5 X 105 ~ 1.3 X 107. KLH is usually associated
with divalent cations. Unlike other gastropod hemocyanins, this protein does not
dissociate simply by removing divalent cations from a hemocyanin suspension,
although divalent cations aid in the formation of larger aggregates.
Aggregation
KLH
exists in five different aggregate states in Tris buffer, pH7.4. With moderate
changes in pH, it will reversibly dissociate to subunits, and at pH8.9 will
completely dissociate. Each subunit contains oxygen binding sites and for every
two atoms of copper in KLH, one molecule of oxygen can be found. The
oxygen-containing KLH is blue, while the oxygen-lacking from is colorless.
Removal of oxygen will also dissociate the protein to lower aggregate states.
Because of increased availability of antigenic sites, increased antibody binding
can be expected when KLH is dissociated into subunits.
Solubility
Solubility in aqueous
solution: Because of its size, KLH often suffers from poor water solubility.
While this may not affect its immunogenicity, it causes the difficulty in
handling of KLH in solution and affects the process of conjugation. Even
following removal of insoluble particles from a KLH solution, it is difficult to
determine the amount of KLH present. KLH solution are turbid so 280 nm
absorbance readings are inaccurate.
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