With the purpose of identifying the Zn²⁺–binding sites in factor XII, the effect of chemical modification of His, Glu and Asp residues, amino acids known to participate in the catalytic coordination binding of Zn²⁺ in a number of Zn²⁺–binding proteins, was analysed. The number of modifiable His residues in factor XII and α–factor XIIa was 16.0±0.7 and 17.3±0.7, respectively. When factor XII/α–factor XIIa was incubated with saturating concentrations of Zn²⁺ before the diethylpyrocarbonate modification of the His residues, these numbers were reduced to 6.3±0.1 and 8.2±0.5, indicating that ten and nine His residues, respectively, are involved in the binding.

Analysis of the Zn²⁺–binding capacity of factor XII, α–factor XIIa and β–factor XIIa showed that while factor XII contains four Zn²⁺–binding sites, α–factor XIIa had only three and β–factor XIIa had none. Modification of the His residues resulted in a complete loss of Zn²⁺‐binding while Asp/Glu modification resulted in loss of two and one Zn²⁺‐binding sites in factor XII and α‐factor XIIa, respectively. This suggests that two of the four sites in factor XII contain His residues, exclusively, while the two others are comprised of two His residues and one Asp/Glu residue. One of the latter is lost when factor XII is activated to α‐factor XIIa. Two of the sites are suggested to be located at positions His40‐His44 and His78–His82. The location of the remaining two sites are reduced to four possible positions.