The purification of human proteolytic enzymes, i.e. two trypsins, two chymotrypsins and a t least one elastase from activated extracts of human pancreas has been accomplished by a combination of a,ffinity chromatography and conventional ion-exchange chromatography on XE-Xephadex. The enzymes thus obtained were homogeneous by several criteria : gel electrophoresis, equilibrium sedimentation and immunological assay. The molecular weight and amino-acid composition of each preparation was determined and compared with those of other mammalian enzymes. The two trypsins showed only partial immunological identity while the two chymotrypsins were identical immunologically. The data raises the question of the number of the zymogens present in the human pancreas.I n addition to the proteolytic enzymes, four protein inhibitors of proteolytic enzymes were also isolated from human pancreas. The procedure consisted of several steps that included gel filtration, affinity chromatography and isoelectric focusing. The two major components which accounted for most of the trypsin inhibitory activity were characterized and were apparently of the Kazal type. The more basic human pancreatic inhibitor had a p I of 8.7 and contained 48 amino acids with a molecular weight of 5200. The more acidic inhibitor had a PI of 6.5 and contained 39 amino acids with a molecular weight of 4300. Both inhibitors were capable of inhibiting bovine and two human trypsins. They did not inhibit two human chymotrypsins but had a weak capacity to inhibit bovine chymotrypsin.A large body of information has been gathered during the past decade concerning the enzymatic composition of pancreatic juices from various mammalian sources. However, there are only a few reports describing the composition and properties of human pancreatic proteins. This is mainly due to the scarcity of human pancreas and to the difficulties arising from the autolysis of the proteases.Buck et al. Abbreviations. EAhx-Trp-OMe, e-aminocaproyl-mtryptophan methyl ester ; Tos-Arg-OMe, N-p-tosyl-L-argininemethyl ester; Ac-Tyr-OEt, N-acetyl-L-tyrosine ethyl ester; Tos-Phe-CH,CI, ~-l-tosylamido-2-phenylethylchloromethylketone; Tos-Lys-CH,Cl, l-chloro-3-tosylamido-7-amino-2-heptanonc ; Dip-I?, di-isopropylfluorophosphate.Enzymes. Trypsin (EC 3.4.4.4) ; chymotrypsin (EC 3.4.4.5); elastase (ED 3.4.4.7).Definition. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 a t 280 nm when measured in a 1-cm pathlength -cell.trypsin. Silberberg and Hadron [3] also reported the presence of two chymotrypsin isoenzymes, while Coan and Travis [5,6] found the presence of three forms of chymotrypsin. Differences in the isolation procedures employed, as well as the inherent autodigestabiliby of the pancreatic proteases, seem to be the main reason for these conflicting reports. The work presented here describes a method for the simultaneous isolation of human pancreatic enzymes : trypsin, chymotrypsin and elastase from frozen whole pancreas. The purification schemes were ...