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Synopsis: 1. A detailed study of the denaturation and renaturation of native soluble creatine kinase (EC 2.7.3.2) has revealed that denaturation by guanidine hydrochloride is essentially completely reversible with 95% regain of enzyme activity after 1.0 h of renaturation at 20°C. In addition the product of renaturation, the fully renatured enzyme, was found to be identical to the native soluble enzyme in terms of active site structure, homogeneity and overall conformation. 2. The dimeric enzyme was immobilised via a single subunit to CNBr-activated Sepharose 4B to yield a matrix-bound enzyme derivative containing 400 µg of protein/ml of packed gel, with substantial retention of enzyme activity. The matrix-bound enzyme was subsequently treated with guanidine hydrochloride to facilitate dissociation of the subunits, followed by renaturation to yield a catalytically active matrix-bound subunit derivative containing 200 µg of protein/ml of packed gel. 3. The effects of pH on the enzyme activities of the soluble enzyme and the matrix-bound derivatives were studied. In all cases there was a broad peak in the pH-activity profile with maximum activity being observed at pH 9.0. Kinetic parameters for the matrix-bound derivatives were generally similar to those for the soluble enzyme although some variations in the values of the Km for MgATP were observed with those for the matrix-bound derivatives being raised approximately 2.5-fold compared with the soluble enzyme. 4. In common with the findings of studies on glucose 6-phosphate dehydrogenase and tryptophanase by other workers (see Chapter 6), it was found that immobilisation of creatine kinase led to an enhancement of stability towards either thermal inactivation or unfolding by guanidine hydrochloride. In both types of study the order of stabilities observed was matrix-bound dimeric enzyme > matrix-bound subunit enzyme > soluble enzyme. 5. The numbers of reactive thiol groups in the soluble enzyme and the matrix-bound derivatives were determined by two different methods: (i) spectrophotometrically using 5,5'- dithiobis(2 -nitrobenzoic acid); (ii) reaction with radioactive iodoacetamide. The results of these two studies indicated that both the soluble enzyme and the matrix-bound dimeric enzyme possess one reactive thiol group per subunit, whereas the matrix-bound subunit enzyme was found to possess two reactive thiol groups per subunit. Further studies on the reactivity of the thiol groups of the soluble enzyme and the matrix-bound derivatives were obtained by measuring the rate of inactivation of the enzyme by iodoacetamide. The data obtained showed that the reactivity of the thiol groups in the presence of various combinations of substrates and ligands of the soluble enzyme and the matrix-bound derivatives were largely similar. 6. The remarkable similarity in the properties of the native and immobilised creatine kinase attests to the lack of significant conformational restraints imposed by the matrix.