The acid phosphatase of bovine milk was further purified to yield enzyme with an activity of about 2 units/mg. This was almost 105 times the activity present in milk and enabled a detailed study of heat inactivation to be made, together with further measurements on binding to casein substrates.

The effectiveness of caseins as inhibitors of the hydrolysis of p-nitrophenyl phosphate by acid phosphatase paralleled the phosphate content of the casein molecules, so that αs1-casein A was a more potent inhibitor with a K1 of 1·7 mM than β-casein A1A2 (K1 = 4·3 mM), which in turn was more inhibitory than κ-casein A (K1 = 5·9 mM).

The heat inactivation of acid phosphatase followed first-order kinetics at pH 4·9, 5·2 and 6·7 and values of E, the activation energy, were between 2·4×105 and 3·0×105 J mole −1 in all cases, consistent with simple protein denaturation. The presence of 1% αs1-casein A, 1% β-casein A1A2, 1% κ-casein A, 1% isoelectrically precipitated ‘whole’ casein and 1% fresh raw milk provided no substrate protection at pH 5·2 or 6·7. Acid phosphatase was somewhat less heat stable at pH 6·7 than at pH 4·9, but may be expected to survive typical milk pasteurization conditions almost completely. However, conventional milk sterilization or ultra-high-temperature (UHT) processes would be expected to give total inactivation.