Effect of dodecylsulfate on trypsin and alpha-chymotrypsin. II. Characterization of soluble combinations

Abstract

The viscosimetric and electrophoretic behaviour of Solutions containing both sodium dodecylsulphate (SDS) and either trypsin or a-chymotriypsin, suggest the production of two different complexes: one of them seems to be formed when the relationship SDS/Protein cquals 0.2 (corresponding to about 20 mols DS- per mol of protein), the second complex, when the valué is higher, up to 0.4 (40 mols DS-). From these soluble, DS-Protein com­plexes, the corresponding free proteins can be recovered either by means of a DEAE chomatographic separation, or by adding Ba" to the Solutions, when all the DS- comes down while liberating the attached proteins. By a careful elution of the DEAE adsorbates, two dif­ ferent fractions are recovered : the one which comes out first from the colunin is enzimatically active, whereas the se­ cond fraction is inactive (probably the denatured protein). The relative proportion of the first, active fraction, diminishes as the relation ship SDS/Protein increases, or the detergent and protein contact period is longer. When the SDS/Protein valué is higher than 1.4, only inactive trypsin is recovered; with a-chymotrypsin, no activity is recovered when the valué equals 2. With casein as a substrate, SDS pro­ ves to be a competitive inhibitor of trypsin and a-chymotrypsin proteolytic activities. In the case of trysin the value of Ki(SDS) = 2.34 x 10-5 M the corresponding value established with a-chymo- trypsin is Ki(SDS) = 1.5 x io"s M. SDS denatures trypsin and a-chynio- trypsin in a process that seems to be a function of the SDS relative concentra­tion and of the contact time between the detergent and the proteins.