[Protaminase activity of plasma. III. Role of conversion enzyme (kininase II) in protaminase activity of plasma]. / Activité protaminasique du plasma. III. Rôle de l'enzyme de conversion (kininase II) dans l'activité protaminasique du plasma.

Kininase I (carboxypeptidase N) and kininase II (angiotensin converting enzyme) were isolated from human plasma by gel filtration on Sephadex G 200, then separated and partially purified by ion exchange chromatography. These two partially purified enzymic preparations allowed us to demonstrate that protamine underwent an extensive degradation only when both kininases acted simultaneously. The effects of CoCl2, an activator, and of several inhibitors, amongst which captopril, suggest that the same enzymatic system is responsible for the in vitro protaminasic activity of diluted unfractionated plasma.

Determination of "in vitro" degradation of protamine in plasma by three different methods.

Three techniques for the quantitative or semi-quantitative determination of the degradation of protamine in plasma are described. One is based on the measurement of liberated arginine, since arginine is the single most important constituent of protamine (80% in weight). The second utilizes successive estimations of protamine by addition to a secondary heparinized medium in which excess heparin is measured by thrombin time and polybrene titration. The third method employs electrophoresis on cellulose acetate, and offers direct visualization of the soluble complexes formed between protamine and albumin, and of their degradation. When applied to an incubation mixture containing diluted plasma (1 : 8) and protamine 0.8 mg/ml, the first two methods were well correlated and showed that protamine degradation proceeded linearly with time. The third method had good semiquantitative agreement with the two former. The rate of protamine degradation was different when estimated by each of the three methods, due probably to the different physico-chemical reactions involved.

[Protaminase activity of plasma and serum in vitro. II.- Electrophoretic study of serum albumin-protamine soluble complexes]. / Activité protaminasique du plasma et du sérum in vitro. II.- Etude électrophorétique des complexes solubles sérumalbumine-protamine.

Cellulose acetate electrophoresis (pH 8,6 ionic strength 0,05) puts in evidence the soluble complexes "albumin-protamine" which are easily distinguished from albumin being more positively charged. The albumin-protamine complexes formed in serum or plasma, after addition of protamine, undergo in vitro a dissociation progressing with time to the complete restitution of albumin. This dissociation is slowed down by the inhibitors of the carboxypeptidase B (SCPB), an enzyme present in plasma and serum, but is not influenced by the inhibitor phenylmethyl-sulfonyl fluoride (PMSF). A protamine which had lost its four C-terminal arginines by the action of a DFP-treated carboxypeptidase B (CPB) still formed complexes with albumin (and, besides, remained able to neutralize heparin). On the contrary protamine degraded first by CPB, and afterwards by the DFP-treated carboxypeptidase A (CPA) lost these two properties. These results suggest that the dissociation of albumin-protamine complex in plasma and serum requires a protaminase action additional to the action of SCPB.

["In vitro" protaminase activity of human plasma and serum and the human carboxypeptidase N (author's transl)]. / Activité protaminasique du plasma et du sérum humains "in vitro" et la carboxypeptidase N.

1 - Protamine was incubated "in vitro", with human plasma and serum, and the initial degradation products (1-4 hours at 30 degrees C) were characterized by paper chromatographic and paper electrophoretic methods. The analysis revealed the presence of the arginine and also, in low quantities, that of a supplementary compound, an arginine peptide. Comparative experiences with the same substrate revealed that carboxypeptidase B liberated exclusively the C-terminal arginine, plasmin exclusively peptides of arginine, and trypsin, besides arginine peptides, minutes quantities of free arginine. 2 - The method used for quantitative determination of arginine liberated by the action of plasma and serum is based on coprecipitation of residual and partly degradated protamine with the plasma proteins by addition of methanol-acetone. The arginine recovered from supernatant is determined with the Sakagushi reagent. 3 - We could thus demonstrate that the protaminase activity of human plasma and serum in greatly enhanced by cobalt and is inhibited by cadmium and EDTA. Trypsin inhibitor of Künitz and PMSF have little effect. These characters are those of the human carboxypeptidase N of Erdös and al. 4 - The comparison of protamines from different laboratories did not reveal essential differences between protamine sulfate and protamine chloride as some authors have claimed. 5 - The results of this investigation suggest that the "in vitro" enzymatic action of human plasma and serum on protamine is mostly due, at least during the first hours of incubation, to the carboxypeptidase N, described by Erdös and al. A low enzymatic additional action on protamine was observed. This action of proteasic character was not inhibited by the trypsin inhibitor of Künitz.