The spontaneous breakdown of human fibrinogen or the lysis of Laki's fibrinogen by purified human fibrinolysin and streptokinase involves at least two different reactions. Clottability by thrombin and precipitability at 25% ammonium sulfate saturation are gradually lost but although clottability disappears completely at a fairly early stage, precipitability is still retained by 4–12% of the original protein even during advanced stages. Both the amount and the antithrombic activity of the anticoagulant fraction of incubated fibrinogen (AFIF) produced vary throughout fibrinogenolysis. The changing quality of AFIF is further evidenced by changes in the electrophoretic patterns which indicate variations in concentration, mobility, and number of components. Strip electrophoresis of fractions isolated by continuous-flow paper electrophoresis showed the presence of two components in the largest fraction but only of a single one of different mobility in a second, smaller fraction. The last finding makes it unlikely that the antithrombic effect exerted by the latter fraction could be due to contamination by components of the former.

Anticoagulant fraction of incubated fibrinogen (AFIF), obtained from human fibrinogen by simple incubation or from bovine fibrinogen (prepared by the method of Laki) after addition of streptokinase-activated human fibrinolysin and incubation for several days, was subjected to continuous flow paper electrophoresis in 0.02 m, pH 8.6 Veronal buffer, at 800 v and about 40 ma. Both kinds of AFIF separated into two components: a major one having the same electrophoretic mobility as the intact fibrinogen and a second more electronegative amounting up to 21% of the first. The latter migrated with a mobility intermediate between that of albumin and α-globulins or between the mobilities of α-and ß-globulin fractions of human plasma, depending on the origin of the preparation human or bovine, respectively. Further studies showed that if the incubation of human fibrinogen was interrupted before the sample became completely unclottable by thrombin, the AFIF obtained showed only the first component on electrophoresis. Both fractions of human AFIF were found able to inhibit the clotting of bovine fibrinogen by thrombin to the same extent as the original sample, however, only the second component inhibited the rate of formation of plasma thromboplastin when added to a thromboplastin generating system.

1. The amino acid compositions of human fibrinogen and three intermediate anticoagulant derivatives were determined by column chromatography. The derivatives were isolated by ammonium sulphate fractionation and column electrophoresis from solutions of fibrinogen undergoing spontaneous breakdown. One derivative, isolated as the large electrophoretic peak at the end of the clottable period (100% CP) of the parent fibrinogen solution, was labelled LP(100) and others obtained at twice this period (200% CP) were designated as LP(200) and SP(200) (LP, large peak; SP, small peak). 2. Maximal ;molecular' weights of approx. 294000 for LP(100), 137000 for LP(200) and 37000 for SP(200) were calculated for the protein moieties. At least 265 amino acid residues must have been lost from each fibrinogen molecule during the formation of LP(100), and 1362 during the formation of the other two derivatives. 3. Only one derivative (LP(200)) had a partial specific volume ([unk] 0.725ml./g.) different from that of fibrinogen ([unk] 0.721ml./g.). 4. No significant differences in refractive index at 589mmu were detected. 5. Calculation of the total number of ionizable groups/10(5)g. of each protein moiety showed a preponderance of the following numbers of negative charges: 22 in fibrinogen; 24 in LP(100); 26 in LP(200); 49 in SP(200). The isoionic points were estimated to be approx.+0.03pH unit (for fibrinogen), -0.06pH unit for (LP(100)) and +0.28pH unit (for LP(200)) from the pK of imidazole, and 0.78pH unit above the average pK of aspartyl and glutamyl ions (for SP(200)). These figures agree closely with experimentally determined values of the isoelectric point of fibrinogen and its derivatives.

Alkaline phosphatase activity was demonstrated throughout the entire length of the small intestine of the albino rat and the relative amounts present seemed to decrease exponentially from the pylorus to the ileocolic valve. All subcellular fractions of intestinal homogenates were found to hydrolyze sodium β-glycero-phosphate. The distribution of activity of the enzyme was as follows: "microsomes", about 76%; nuclei, 10%; mitochondria, 8%; and in the nongranular, supernatant fraction, 9%. The specific activity for these fractions was: 52, 3, 10, and 3, respectively. The effects of the time of incubation, pH of the hydrolytic mixture, and the concentration of the enzyme were similar in all fractions. For the enzyme in the various fractions slight differences were observed in the values of the Michaelis constants, and in the degree of activation by magnesium ion. These variations may be explained on the basis of differences in the accessibility of substrate and magnesium ion to the enzymes in the various fractions. The finding that the "microsomes" contained the highest levels of alkaline phosphatase suggests that this intestinal enzyme is produced almost entirely by these submicroscopic particles.

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