Single-chain Species Research Articles

Exploring the Growth Dynamics of Size-Selected Carbon Atomic Wires with In Situ UV Resonance Raman Spectroscopy.

Short carbon atomic wires, the prototypes of the lacking carbon allotrope carbyne, represent the fundamental 1D system and the first stage in carbon nanostructure growth, which still exhibits many open points regarding their growth and stability. An in situ UV resonance Raman approach is introduced for real-time monitoring of the growth of carbon atomic wires during pulsed laser ablation in liquid without perturbing the synthesis environment. Single-chain species' growth dynamics are tracked, achieving size selectivity by exploiting the peculiar optoelectronic properties of carbon wires and the tunability of synchrotron radiation. Diverse solvents are systematically explored, finding size- and solvent-dependent production rates linked to the solvent's C/H ratio and carbonization tendency. Carbon atomic wires' growth dynamics reveal a complex interplay between formation and degradation, leading to an equilibrium. Water, lacking in carbon atoms and reduced polyynes solubility, yields fewer wires with rapid saturation. Organic solvents exhibit enhanced productivity and near-linear growth, attributed to additional carbon from solvent dissociation and low relative polarity. Exploring the dynamics of the saturation regime provides new insights into advancing carbon atomic wires synthesis via PLAL. Understanding carbon atomic wires' growth dynamics can contribute to optimizing PLAL processes for nanomaterial synthesis.

PH-Sensitive morphological transitions in polymeric tadpole assemblies for programmed tumor therapy

Ultrafine single-chain tadpole polymers (SCTPs), containing an intrachain crosslinked globule and a pH-sensitive linear polymer chain, have been synthesized. Self-assembly of these polymers depends on the linear block length and the pH, at which the polymer is assembled. Although the SCTPs themselves exhibit a size that is consistent with a single-chain species, the self-assembled SCTPs were found to be substantially larger. Since the transition between these two structures is reversibly dependent on pH, we explored the possibility of utilizing these assemblies to achieve deep tissue penetration in tumors. Our results indicate that there is indeed a pH-dependent deep tissue penetration in ex vivo tumor multicellular spheroids. Moreover, the multi-tadpole assemblies (MTAs) can stably encapsulate hydrophobic molecules, which have been used to encapsulate paclitaxel (PTX). These PTX/MTAs show excellent therapeutic efficacy and biosafety in 4 T1 xenograft mouse models. The innovative multi-compartment aggregates are able to fulfill structure-related function transitions with the variation of microenvironment, which has potential to extremely enrich the design of sophisticated biological agents.

Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses

One of the β-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-β-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-β-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 106 M−1. The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-β-glucanase.

Titration of DNA/Carbon Nanotube Complexes with Double-Chained Oppositely Charged Surfactants.

1/1 dispersions of ss-DNA/CNT complexes in mass ratios were investigated in a mixture with didodecyldimethylammonium bromide, DDAB. Depending on the amounts of the surface-active agent and of the complexes, solutions, precipitates, or re-dissolution occur. DDAB titrates the phosphate groups on the outer surface of the complex and controls the phase sequence in these systems. The combination of different experimental methods determined the phases that occur therein. The results are based on optical absorbance, Dynamic Light Scattering, ionic conductivity, ζ-potential, optical microscopy and AFM. From the above findings a (pseudo)-binary phase diagram is attained. The system has strong similarities with polymer-surfactant mixtures. In fact, its properties conform to cases in which interactions between rigid rod-like polyelectrolytes and oppositely charged species take place. The peculiarities of double-chained DDAB in the process imply significant differences with respect to the behavior of single chain surfactants. In fact, DDAB associates into vesicular entities, when the homologous single chain species forms small micellar aggregates.

Macromolecular and Morphological Evolution of Poly(styrene sulfonate) Complexes with Tetradecyltrimethylammonium Bromide

Macromolecular characteristics and morphology of water-soluble complexes between sodium poly(styrene sulfonate) (PSS) and tetradecyltrimethylammonium bromide have been followed as a function of surfactant-to-polymer charge ratio (S/P) to elicit possible changes in the complexation mechanism. As revealed by light scattering, shorter PSS (30 and 150 repeat units) yield multichain complexes while longer PSS (450 and 5000 repeat units) form single-chain species throughout 0 < S/P < 0.9. Irrespective of PSS chain length, the complexes exist in solution in a swollen coil conformation and undergo a compaction with S/P but never collapse into a globule. Even when the free PSS chain is too short to coil (30 repeat units), the complexes adopt a coiled conformation due to multichain aggregation. Morphological changes (manifested by a hypochromic shift in UV spectra of the complexes at S/P < 0.5 and an increase in the local surfactant mobility observed at S/P > 0.5 by ESR) strongly suggest a change in the formation mechanism of the complexes with a transition near S/P = 0.5.

Increased Stability and DNA Site Discrimination of “Single Chain” Variants of the Dimeric β-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric beta-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

Effect of electric field, solvent, and concentration on the electroluminescence spectra and performance of poly[2-methoxy-5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene] based light emitting diodes

The electroluminescence (EL) spectra and performance of polymer light emitting diodes based on poly[2-methoxy-5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene] are found to depend significantly on the applied electric field as well as the solvent and concentration used in the fabrication of the polymer layer. Drastic changes in the EL spectra with the electric field are observed in devices in which the polymer layer is spin cast from chlorobenzene (CB) at high concentration. At low electric field, the broad reddish-orange emission from these devices originates mostly from aggregate species formed by interchain interactions. However, at high electric field, a narrow yellow emission is observed, which originates from both aggregate species (minor part) as well as single-chain species (major part) arising out of intrachain interactions. The individual emission intensities of the two species are estimated at different electric fields and are found to behave in a different manner with the change in electric field. The fractional contribution of the aggregate species in the overall EL spectra changes from 98% to 17% as the electric field is increased from low to a high value. Either changing the solvent from CB to tetrahydrofuran (THF) or decreasing the concentration (in any particular solvent) reduces the possibility of the formation of aggregate species. At any particular concentration, the turn-on field is found to be always higher in CB-cast films than THF-cast ones. This fact is explained by the presence of deeply trapped holes at low electric field. Devices made from CB are found to give better performance regarding external quantum efficiency, power efficiency, etc., than those made from THF, irrespective of the concentration. Again, devices made from any particular solvent are found to perform better at low concentration than at high concentration. Enhanced carrier mobility accounts for the improved performance of the devices made from CB than those made from THF. On the other hand, device performance becomes poor at high concentration (in any particular solvent) due to the formation of aggregate species. The optimum performance is obtained from the devices in which the polymer layer is spin cast from CB at low concentration.

Separation of ergot alkaloids by micellar electrokinetic capillary chromatography using cationic Gemini surfactants

A micellar electrokinetic capillary chromatographic method using novel cationic dimeric (Gemini) surfactants has been developed for the separation, in less than 8 min, of 17 dihydroergotoxines, aci-alkaloids and oxidation products. Both 1,3-bis(dodecyl- N, N-dimethyl ammonium)-2-propanol (20 m M) and 1,3-bis(tetradecyl- N, N-dimethyl ammonium)-2-propanol (40 m M) surfactants in 50 m M phosphate buffer, pH 3.0, at 20°C produced separations that could not be achieved using single chain tetradecyl- or hexadecyl-trimethyl ammonium micelles. The retention factors ( k′) increased linearly with surfactant concentration in accordance with theory. Similarly, the net mobilities generally decreased nonlinearly with surfactant concentration. The smallest alkaloids, i.e. the 6-methylergoline-8-carboxylic acids, and the more polar aci-alkaloids with a Z-type conformation, have the smallest k′ values. For the larger alkaloids, the k′ values with the Gemini surfactants were smaller than with the single chain species, possibly because the more rigid dimeric molecules hinder the partitioning of the alkaloids into the micelle as well as the greater hydrophilicity of the hydroxylated micelles. Increasing buffer pH mainly affects the more strongly acidic and basic alkaloids, causing a marked increase and decrease in net mobilities, respectively. k′ values generally increased with phosphate buffer concentration because of the decreasing solubility of the hydrophobic alkaloids with increasing ionic strength.

Factors X Wenatchee I and II: compound heterozygosity involving two variant proteins

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10–20% of normal) and antigen (30–35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13 984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors X Wenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

Preclinical Studies and Optimization of Specific Tumor Delivery for 741F8 sFv, an Anti-c-erbB-2 Single-Chain Species

AbstractSingle-chain Fv fragments (sFv) that bind tumor-associated antigens exhibit highly specific tumor targeting characteristics, based on studies of intravenous sFv administration to immunodeficient mice bearing human tumor xenografts. These features suggest that sFv may be used as targeting vehicles for diverse agents such as radionuclides, toxins, chemotherapeutic agents, or genes. However, the quantitative tumor retention of sFv molecules is lowered by their fast clearance and rapid dissociation from antigen. We have found that the retention of tumor-specific 74IF8 sFv in tumor is an antigen-specific event and not the result of extravascular pooling. We also have produced divalent 741F8 (sFv')2 species and have examined their pharmacokinetics and biodistribution properties in relevant tumor-bearing scid mice. Finally, we have employed a number of strategies to optimize the tumor-specific retention of radiolabeled sFv molecules; escalations in administered doses, repetitive intravenous bolus adminis...

Comparison of surface properties and thermodynamic behavior of lyso- and diacylphosphatidylcholines

Critical micelle concentrations as a function of temperature have been determined by surface tension for a series of diacylphosphatidylcholines and lysophosphatidylcholines with the same total number of fatty acyl carbons. From these studies, thermodynamic functions (Δ G mic, Δ G ads) and various interfacial parameters (surface areas and pressures) are derived and used to compare the double- and single-chain species. Key conclusions are that (i) micellization is more dependent on hydrophobic interactions than adsorption, (ii) four carbons contribute to the micellization process but do not contribute to adsorption and this can be related to hydration of the lipids, and (iii) chain length limits can be set on which lipids no longer form micelles or adsorb to the air/water interface.

Melting of myosin and tropomyosin: Electron microscopic observations

A method was devised to maintain a very low angle (2–3°) during the metal casting of specimens for electron microscopy. With this modified rotary shadowing procedure the melting of myosin and tropomyosin (TM) was investigated. When protein solutions were sprayed on mica sheets and then heated to melt α-helices, myosin molecules did not show any sign of chain separation but appeared to have collapsed into loose clumps. A few molecules showed separation of the two chains at the light meromyosin-heavy meromyosin hinge region. Heating myosin in bulk solution at 65°C before spraying caused extensive fusing of the myosin heads. In contrast, in the case of TM, separation of the chains appeared to occur at temperatures at which the unfolding of α-helices had been shown by circular dichroism. Dissolution of TM and myosin in 0.5% SDS followed by 150-fold dilution led to single chain species. This method capable of detecting single chain peptides of melting TM whose thickness is of the order of 1 nm may be applicable to the study of the structure of proteins previously not considered possible.

Multiple Domains of Botulinum Neurotoxin Contribute to Its Inhibition of Transmitter Release in Aplysia Neurons

The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.

A human hepatoma cell line expresses a single-chain form of gamma-glutamyl transpeptidase.

Mammalian gamma-glutamyl transpeptidases characterized thus far have been shown to be heterodimeric glycoproteins. The two subunits are derived from a single-chain propeptide which, in the rat kidney, exhibits low transpeptidase activity (less than 2% of the dimeric enzyme). A human hepatoma-derived cell line, Hep G2, expresses relatively high transpeptidase activity. The enzyme is primarily localized on the cell surface and exhibits catalytic properties similar to the dimeric human kidney and lymphoid cell transpeptidase. Significantly, the Hep G2 enzyme, unlike the enzyme from other human tissues, is a single-chain species, Mr = 120,000.

Activity measurements and spectroscopic studies of the catalytic oxidation of toluene over V2O5/Al2O3-C catalysts

Vanadium oxide catalysts based on Al2O3-C (γ-Al2O3) as support contain at least four different forms of vanadium. The distribution between these depends on the vanadium loading. The activity (per gram of vanadium) of these catalysts for toluene oxidation increases with increasing loading. The selectivity for benzene formation decreases from 2% for the support to 0% for 0.5 wt% V, while the selectivity for benzaldehyde formation first appears at this concentration and rises to 29% for 10 wt% V. It is suggested that benzene is formed at Lewis acid sites on the support, whereas benzaldehyde is formed on vanadium sites. At low loadings (0.1 and 0.2 wt% V) single vandium species with tetrahedral coordination are formed. The oxidised forms have u.v. bands at 35 500 and 42000 cm–1 and the reduced forms have i.r. bands of adsorbed CO at 2200 cm–1(room temperature) and at 2190 and 2158 cm–1(133 K). At medium loadings, vanadium surface clusters with varying degrees of agglomeration are formed in addition to the other species. These are suggested to be single chain species with vanadium in tetrahedral coordination, double chain species with vanadium in square pyramidal coordination and aggregates of octahedral vanadium formed by the coupling of double chains. The oxidised forms have u.v. bands at 34 500 and 43000 cm–1 and the reduced forms have i.r. bands of adsorbed CO at 2178 cm–1(room temperature) and at 2178 and 2158 cm–1(133 K). The agglomerates are more active than the isolated species and show some selectivity for benzaldehyde. Both the activity and the selectivity appear to increase with the degree of agglomeration. At high vanadium loadings (10 wt% V), surface crystallites of vanadium oxide are formed in addition to the other species. The oxidised forms have u.v. bands at 30 000 and 43 000 cm–1 and the reduced forms have i.r. bands of adsorbed CO at 2181 cm–1(room temperature) and at 2181 and 2145 cm–1(133 K). These crystallites are more active and selective than other species with less agglomeration. It is suggested that the increased activity for the larger species is due to the possibility of a transition from corner to edge sharing octahedra at the release of oxygen, which increases the activity of the double-bonded oxygen.

Double-Stranded Helix of Xanthan: Dissociation Behavior in Mixtures of Water and Cadoxen

A sodium salt xanthan sample dissolved directly in mixtures of water and cadoxen was investigated by elastic light scattering and viscometry at 25°C. With the increase in weight fraction wcad of cadoxen in the mixed solvent, the weight-average molecular weight, z-average radius of gyration ‹S2›z1/2, and intrinsic viscosity [η] of the sample fairly sharply decreased from the values for the double-helical dimer (in 0.1 M aqueous NaCl) to those for the single random coil (in pure cadoxen) in a range of wcad from 0.3 to 0.8. It was found that these data can be explained if it is assumed that only these two species coexist in the mixed solvent and if care is taken of the fact, found from measurements on cadoxen solutions diluted with water, that ‹S2›z and [η] of the single chain species markedly increase with decreasing wcad from 1.0 to 0.6. Thus, it was concluded that xanthan double helices in water-cadoxen mixtures dissociate into single chains directly, i.e., without passing through their partial melting. The expansion of the single chain species was attributed to the formation of intramolecular hydrogen bonds.

Turnover of heparan sulfate proteoglycans from substratum adhesion sites of murine fibroblasts.

Substratum adhesion sites from murine Balb/c SVT2 fibroblasts are enriched in heparan sulfate proteoglycans which have been implicated in mediating adhesion of these cells to a fibronectin-adsorbed tissue culture substratum. Most of the heparan sulfate isolated from newly formed adhesion sites is found covalently attached to protein as proteoglycan while a significant portion of heparan sulfate from older sites has been identified as a single-chain species. This observation suggests that there may be catabolism of the heparan sulfate proteoglycan during the "maturation" of these adhesion sites at the cell's undersurface. Zwittergent 3-12 selectively extracts the single-chain class of heparan sulfate from either newly formed or "mature" adhesion sites while leaving the proteoglycan firmly bound in these sites. In an effort to further characterize the metabolism of these proteoglycans, substratum adhesion sites were isolated at various times after the cells had been pulse-radiolabeled using radioactive sulfate and subsequently chased. Greater than 80% of the sulfate-radiolabeled material is lost from the substratum-attached material within 24-48 h. Characterization of both the Zwittergent-soluble and -resistant heparan sulfate indicated that there was an initial accumulation followed by a rapid loss of a portion of the radiolabeled heparan sulfate as the single-chain Zwittergent-soluble class. However, most of the heparan sulfate proteoglycan was lost from the adhesion sites following approximately a 4-h time lag during the chase period without going through a smaller molecular weight intermediate. The turnover properties of the heparan sulfate proteoglycan in the EGTA-detachable cells were different from those in the substratum-attached fraction of the cell. The significance of these two different mechanisms of turnover of heparan sulfate proteoglycan in adhesion sites is discussed in relation to the role of this proteoglycan in mediating adhesion processes.

An improved method of isolation of rat pancreatic prokallikrein: Characterization of the zymogen and of the kallikrein produced by trypsin activation

A prokallikrein was purified 1600-fold from rat pancreatic tissue in an overall yield of 40% by a simple four-stage procedure. The final and crucial step was immunoaffinity chromotography utilizing antibody raised to a very small amount of prokallikrein. Both the pure zymogen and the active kallikrein generated from it by trypsin activation are single chain species with M r values of 38 400±300 and 35 500±400, respectively. Valine is the N-terminal amino acid residue of prokallikrein. The zymogen Was comparatively stable both to autoactivation and denaturation with respect to temperature and pH. The kallikrein produced by trypsin activation of the zymogen was similar in some of its catalytic properties to the kallikrein purified from autolyzed rat pancreas but the two species differed in their susceptibility to substrate activation.

Studies on the inhibition of ellagic acid-activated Hageman factor (factor XII) and Hageman factor fragments

Hageman factor (HF, factor XII) that has been exposed to Sephadex- ellagic acid gels is a single-chain species (HFea) with amidolytic properties for the synthetic substrate H-D-phenylalanyl-L-pipecolyl-L- arginine p-nitroanilide. Earlier we reported that amidolysis was suppressed by incubation of HFea with specific antiserum. The present study provides additional evidence that the amidolytic properties of preparations of HFea are ascribable to this substance through an examination of a number of protease inhibitors. HFea's amidolytic properties were inhibited by alpha 2-plasmin inhibitor, antithrombin III in the presence of heparin, and Cl esterase inhibitor (Cl-INH). Additionally, it was inhibited by popcorn inhibitor, leupeptin, hexadimethrine bromide, protamine sulfate, dansyl-arginine N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), diisopropylphosphofluoridate (DFP), aprotinin, and at excessively high concentrations, soybean and lima bean trypsin inhibitors. The spectrum of action of agents that did or did not inhibit HFea supports the view that amidolysis by preparations of HFea is attributable to this enzyme. In general, the enzymatically active carboxy-terminal fragment of HF (HFf) was inhibited by the same agents that inhibited HFea, but aprotinin, protamine sulfate and hexadimethrine bromide were more effective against HFf than HFea, while the reverse was true of lima bean trypsin inhibitor.

Studies on the Inhibition of Ellagic Acid-Activated Hageman Factor (Factor XII) and Hageman Factor Fragments

Hageman factor (HF, factor XII) that has been exposed to Sephadex-ellagic acid gels is a single-chain species (HFea) with amidolytic properties for the synthetic substrate H-d-phenylalanyl-l-pipecolyl-l-arginine p-nitroanilide. Earlier we reported that amidolysis was suppressed by incubation of HFea with specific antiserum. The present study provides additional evidence that the amidolytic properties of preparations of HFea are ascribable to this substance through an examination of a number of protease inhibitors. HFea’s amidolytic properties were inhibited by α2-plasmin inhibitor, antithrombin III in the presence of heparin, and Cl esterase inhibitor (Cl-INH). Additionally, it was inhibited by popcorn inhibitor, leupeptin, hexadimethrine bromide, protamine sulfate, dansyl-arginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA), diisopropylphosphofluoridate (DFP), aprotinin, and at excessively high concentrations, soybean and lima bean trypsin inhibitors. The spectrum of action of agents that did or did not inhibit HFea supports the view that amidolysis by preparations of HFea is attributable to this enzyme. In general, the enzymatically active carboxy-terminal fragment of HF (HFf) was inhibited by the same agents that inhibited HFea, but aprotinin, protamine sulfate and hexadimethrine bromide were more effective against HFf than HFea, while the reverse was true of lima bean trypsin inhibitor.