Tangential flow filtration of haptoglobin

Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Hp has various potential therapeutic applications, but it has been mainly studied for treatment of acute hemolytic conditions that can arise from situations such as massive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, radiation injury, and others. Therefore, Hp may also be beneficial during chronic hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp from plasma or plasma fractions. However, none of these methods have exploited the large molecular weight (MW) range distribution of Hp polymers to easily isolate Hp from other plasma proteins. The present study used tangential flow filtration (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as the starting material. After removal of insoluble material from a suspension of FIV paste, the protein mixture was clarified on a 0.2 μm hollow fiber (HF) TFF filter. The clarified protein solution was then bracketed based on protein MW using HF filters with MW cut-offs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp purity of the main bracket was ~75% with a total Hb binding capacity (HbBC) yield of 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions.     

Characterization of peptides and proteins in commercial HSA solutions

HSA solutions account for 14% of the world market for plasma products. Albumin is indicated for reestablishing and maintaining circulatory volume in situations resulting from traumatic shock, surgery, or blood loss. Albumin is also used in extracorporeal liver support devices that perform blood dialysis against this protein. However, the protein composition of therapeutic albumin is only partially known. We performed an exhaustive analysis of albumin composition using a proteomic approach. Low abundance proteins and peptides in these samples were concentrated using a strong anion exchange resin. The absorbed material was eluted with a stepwise gradient of ammonium trifluoroacetate and the protein fraction was digested and analyzed by multidimensional liquid chromatography coupled to ESI‐MS/MS using a linear ion trap. A total of 1219 peptides corresponding to 141 proteins different from albumin were identified with a false discovery rate <1%. Near 50% of these proteins have been described previously as forming part of the albuminome. Some of these proteins are proteases (kallikrein) or protease inhibitors (kininogen and SRPK1) or have relevant functions in cell surface adhesion (selectin, cadherins, and ICAMs) or in immunity and defense (molecules of the complement system and attractin). Characterization of these proteins and peptides is crucial in order to understand the therapeutic and possible deleterious effects of albumin therapies, in which this solution is infused to treat different pathological conditions.     

A simple two-step purification of protease nexin.

This paper describes a simple purification procedure for protease nexin, a serine proteinase inhibitor secreted by cultured human fibroblasts that regulates proteinase activity at and near the cell surface. The first step in the procedure takes advantage of the high-affinity binding of protease nexin to dextran sulphate-Sepharose. This step eliminates the need for prior concentration of the serum-free fibroblast-conditioned medium, since protease nexin binds to the resin in the presence of physiological saline. The use of dextran sulphate also provides an affinity resin with considerably less variability than the heparin-based resins previously used. Final purification to homogeneity involves a combination of DEAE-Sepharose in-line with dextran sulphate-Sepharose to simultaneously purify and concentrate the protein. Purified protease nexin is shown by Ouchterlony analysis and peptide mapping to be immunologically and structurally distinct from antithrombin III and heparin cofactor II, two plasma proteinase inhibitors with similar properties.     

The economic side of blood banking and plasma fractionation.

While cellular components have a relatively short half-life, must be necessarily administered group-specifically and kept available on a round-the-clock basis, all of which add significantly to the basic cost per unit, products isolated from plasma are not handicapped by these disadvantages. Another important advantage for the production of plasma components lies in the fact that the raw material may be also collected through plasmapheresis, a process which allows the collection of significantly greater amounts of plasma from one donor as compared to conventional whole blood collection. Quite understandably, the maintenance of whole blood and cellular component supplies has been left to national and/or non-profit organizations, while commercial firms run a profitable business with the production and distribution of plasma fractions. The method for the selective isolation of plasma fractions developed in our blood transfusion service solves the high cost problems involved in conventional fractionation methods but does not solve the ethical and economical problem related to discarding precious unused raw material.     

Separation of gangliosides on a new type of anion-exchange resin

A new anion exchange resin, Spherosil-DEAE-Dextran, consisting of porous glass beads covered with cross-linked DEAE-Dextran, was used in the separation of gangliosides. The gangliosides were eluted from the resin with a discontinuous gradient of potassium acetate in methanol, separating the gangliosides quantitatively into mono-, di, tri-, tetra- and pentasialoganglioside fractions. The new resin was found to have higher binding capacity, to show less unspecific adsorption and to give a better separation of the higher oligosialogangliosides than did DEA-Spherosil, QAE-Sephadex, DEAE-Sephadex and DEAE-Sepharose. Anion exchange chromatography improved discrimination between closely allied gangliosides as well as quantification and identification of individual gangliosides, especially the minor ones. The new procedure was used in the separation of the gangliosides in human infant forebrain and cerebellum.     

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.     

Partial purification of chlorophyll degrading enzymes from cavendish banana (Musa Cavendishi)

Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30 degrees C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from 'green-ripe' bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from 'green-unripe' and 'yellow-ripe' bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a single major band, resulting in almost a homogenous preparation in a single step. Fraction IV catalyzed dechelation of Mg by Mg-dechelatase, while fraction VI catalyzed the formation of 132-OH-Chl a by chlorophyll oxidase. Chlorophyll oxidase activity was stimulated by linolenic acid, indicating involvement of lipoxygenase in oxidative Chl degradation, thereby resulting in the formation of 13(2)-OH-Chl a as product. The results show the presence of various enzymes of chlorophyllase and chlorophyll oxidase pathways in soluble enzyme fraction.     

In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens

Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to investigate the capacity of these fractions to induce cell proliferation. It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.     

Suproxide dismutase in human testis preparations

A protein fraction from human testis was structurally investigated. The main component of the fraction reported to contain inhibin-like activity was purified and analyzed by tryptic digestion. The peptides obtained identified the protein as an enzyme, superoxide dismutase, previously known to be present in seminal plasma. The results show that superoxide dismutase is a major enzyme, also of testicular material. They further demonstrate the importance of using pure fractions, and controls such as checks with structural analysis or synthetic peptides, in the work of elucidating the nature of inhibin and other hormonal peptides.     

Direct determination of selenium in human blood plasma and seminal plasma by graphite furnace atomic absorption spectrophotometry and clinical application

Direct determination of selenium (Se) in body fluids by graphite furnace atomic absorption spectrophotometry (GFAAS) may suffer from problems like severe background, matrix effects, preatomization losses, and spectral interferences. In this study we evaluate critically the influence on the accuracy of the direct determination of Se in blood plasma and seminal plasma by GFAAS, and propose a simple, rapid, and accurate method, suitable for routine clinical analysis. The method for blood plasma is mainly based on studies by the use of matched matrix and a Pd-Ni modifier, but for seminal plasma only a Pd modifier is required. The method developed was also applied to study the Se distribution in plasma protein fractions of patients with hepatocellular carcinoma. The Se in plasma of patients was significantly lower than that of the controls. The distribution pattern of Se in blood plasma fractions of patients was also different from that of the controls.     

Fractionation of mechanically sheared chromatin on ECTHAM-cellulose

Chromatography of chromatin on the weak ion-exchange resin ECTHAM-cellulose was re-examined using the combined salt-pH elution conditions of Stratling, W.H., Van, N.T. and O'Malley, B.W. (1976) Eur. J. Biochem. 66, 423-433. When mechanically sheared rat liver chromatin was chromatographed on ECTHAM-cellulose the histone composition of eluted fractions was very similar, whereas early eluting fractions were enriched in non-histone proteins, including certain high mobility group proteins, and in hnRNP particles, containing newly synthesised RNA. Later eluting fractions were depleted in all of these components. The majority of hnRNP particles in early eluting chromatin were shown to be physically associated with chromatin by centrifugation in metrizamide. Hen erythrocyte chromatin contained no early eluting material. Size of DNA fragments was not a significant factor in determining the elution position of chromatin fragments. Early eluting material was not generated by endogenous nuclease and protease action. The conditions of chromatin preparation, and of elution of early chromatin fractions caused no gross disruption of chromatin structure, or dissociation of chromatin proteins, although some nucleosome sliding may have occurred. The conditions required for elution of some of the later fractions are sufficient to cause dissociation of protein, and alteration of chromatin conformation.     

Effect of plasma biotinyl-peptides on biotinidase activity

Purified biotinidase (enriched 24,000-fold) from fresh human plasma exhibited reduced catalytic activity when incubated with heat-inactivated dialyzed plasma. The polypeptide fractions separated from the heat-inactivated dialyzed plasma using streptavidin-Sepharose resin showed the same effect on purified biotinidase. These inhibitory effects on biotinidase were partial (25-45%) rather than complete. The polypeptide fraction from streptavidin-Sepharose resin was analyzed by SDS-PAGE in the Laemmli system and by various types of HPLC. Analyses by ion-exchange and reversed-phase HPLC revealed the existence of three relatively small mol. wt polypeptides. Each of these peak fractions exhibited similar inhibitory effects on biotinidase activity. SDS-PAGE analysis indicated that the streptavidin affinity resin fraction was composed of four major polypeptides whose mol. wts were 120,000, 76,000, 53,000 and 27,000. The two bands of 120,000 and 76,000 corresponded to the mol. wts of the biotinyl subunit of pyruvate carboxylase, beta-methyl-crotonyl-CoA and/or propionyl-CoA carboxylase respectively. However, the polypeptides of mol. wts 53,000 and 27,000 were found to be two unique biotinyl-peptides present in human plasma. These bands on the gels were transblotted and exhibited a fluorescent activity after incubated with a FITC-avidin. These findings strongly suggest the existence of circulating plasma biotinyl-polypeptides as inhibitory factor(s) on human plasma biotinidase.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Isolation and partial characterization of glycolipids and a carbohydrate from the secondary granules and plasma membrane of polymorphonuclear leukocytes

Chloroform/methanol extracts of the secondary granule and plasma membrane fractions of polymorphonuclear leukocytes have been shown to contain both non-polar and polar carbohydrate-containing materials. The ratio of the polar to the non-polar material was much higher in the plasma membrane than the secondary granule fraction. The non-polar material contains at least two ceramide-like glycolipids and accounts for most of the broad band of periodic acid/Schiff-positive material which migrates at the dye front in sodium dodecyl sulfate electrophoretic gels of granule and plasma membrane extracts. The polar material appears to be a single substance containing no fatty acids or sialic acid and is composed of glucose, hexosamine and a carboxylic acid derivative of pentose. Expressed on a per mg of protein basis, the amount of carbohydrate associated with the polar material in the plasma membrane fraction was about five times that of the secondary granule fraction.     

The 95000-Mr gelatin-binding protein in human serum and plasma.

A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.     

Mosquito reproduction: Incomplete utilization of the blood meal protein for oögenesis

Fecundity, measured by utilization of dietary protein for the production ofyolk protein was examined in several mosquito species (A. aegypti, A. simpsoni, A. atropalpus, A. triseriatus) using blood from eight vertebrate hosts over a whole range of biological blood mean volumes. All experimental blood meals were injected by enema in measured amounts. The total ovarian and excretory nitrogen produced during each gonotrophic cycle was quantified.Blood proteins are utilized for oögenesis only to a limited extent: two thirds, at the most, are detectable in the mature ovaries. This efficiency decreases with increasing blood meal size to about one fifth. The nitrogen content of each mature egg is constant and species-specific, irrespective of blood meal size or source.There are no significant differences in the efficiency of conversion of protein from different types of vertebrate blood into oöcyte protein except for human blood which led to a drastically reduced efficiency. This is shown to be due to a lack of isoleucine which is essential for reproduction. Hominid primates lack this amino acid in their haemoglobin molecule and it exists at a low titre in human plasma. Apparently, in other vertebrates the amino acids are better balanced in the haemoglobin or, can be mobilized in sufficient amounts from various non-haemoglobin protein fractions.All blood protein is catabolized and the excess nitrogen excreted quantitatively; excretory nitrogen therefore reflects the quantitative demands of a gonotrophic cycle. These processes are not dependent upon receiving a mating stimulus.     

Oral application of insulin encapsulated liposomes

125Iodine labelled insulin encapsulated liposomes were introduced orally to rats. After different intervals of feeding, blood from the portal vein and heart was collected. The plasma was passed through a Sepharose 2B column to establish the presence of liposomes or intact hormone. Liposomal and hormonal fractions from the column loaded with plasma from the portal vain contained radioactivity. It was found that approx. 60% of the loaded radioactivity was associated with the liposomes whereas 20% of the loaded count was found with the hormonal fractions. It is noted that undegraded protein is not detected in portal plasma from rats fed with free insulin. When plasma from the heart of rats fed with liposomal insulin was passed through Sepharose 2B column, neither liposomes nor insulin was detected.     

Characteristics of a new binding protein distinct from the kinase for guanosine 3':5'-monophosphate in rat platelets

A new type of cyclic GMP binding protein was recently identified in our laboratory (Hamet, P. and Coquil, J.-F. (1978) J. Cyclic Nucleotide Res. 4, 281--290). The binding, recovered in the supernatant fractions, is highly specific for cyclic GMP and is clearly distinct from the binding to cyclic GMP-dependent protein kinase. Chromatography on DEAE-Sepharose separated the cyclic GMP binding protein from cyclic AMP binding, cyclic AMP-dependent kinase activities, and from guanylate cyclase. The optimal binding occurs at high pH and in the presence of thiol reagents. Several phosphodiesterase inhibitors increase the affinity of binding (Kd was 353 +/- 60 nM in the absence and 13.4 +/- 1.5 nM in the presence of 1-methyl-3-isobutyl-xanthine). The molecular weight of the binding protein was determined to be about 176,000 and the sedimentation coefficient was 6.4 S. While the binding and phosphodiesterase activities co-migrated on DEAE-Sepharose, gel filtration and sucrose gradients, certain treatments (such as increasing the concentrations of salt and heating) were able to influence one activity while having no effect on the other. Hence, the binding activity may be involved in the regulation of the activity of cyclic GMP phosphodiesterase. Since the binding protein appears to be the only 'receptor' for cyclic GMP detectable in platelets, this protein and/or its relation to cyclic GMP phosphodiesterase may play a role in the mechanism of action of cyclic GMP in platelets.     

Thrombokinase of the Blood as Trypsin-Like Enzyme

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.