Abstract Poly(N-vinylpyrrolidone) (PVP) shielding of Blue Sepharose and Scarlet Sepharose proved to be an efficient method for improving the process of purification of lactate dehydrogenase (LDH) from porcine muscle and secondary alcohol dehydrogenase (SADH) from Thermoanaerobium brockii, respectively. PVP shielding of Blue Sepharose resulted in improvement of the efficiency of either specific or non-specific elution of LDH. PVP protection of Scarlet Sepharose resulted in an improvement in the SADH recovery during specific elution and in an improvement in enzyme purity on non-specific elution. The dynamic capacities of the columns were in both cases decreased after PVP shielding. PVP shielding is considered to prevent the matrices from binding foreign proteins and from non-specific binding of nucleotide-dependent enzymes, while not seriously impairing the specific binding of these enzymes to the affinity matrices.