Hydrophobic Form Research Articles

Heparan sulfate proteoglycans of rat embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components.

Heparan sulfate proteoglycans (HSPGs) synthesized in cultures of rat embryo fibroblasts have been isolated and characterized. Cells grown in the presence of [35S]sulfate secreted a large amount of radiolabeled macromolecules into the culture medium of which only a small proportion was identified as HSPG. However, the majority of radiolabeled proteoglycans isolated from the cell layer were HSPGs. Here, two types of HSPG were detected. One type had an Mr of 5-8 X 10(5) as estimated by gel chromatography on Sepharose CL-4B in the presence of 0.1% sodium dodecyl sulfate and lacked hydrophobic properties in that it showed no affinity for octyl-Sepharose and could not be inserted into liposomes. The other HSPG type had an estimated Mr of 3-5 X 10(5), was retained on octyl-Sepharose, and could be inserted into liposomes. In addition, the cells contained low molecular weight heparan sulfate oligosaccharides. Treatment of living cells with 0.2% Triton X-100, which retained stress fibers and extracellular matrix but solubilized cell membranes, released a proportion of the smaller HSPG together with the heparan sulfate oligosaccharides. The cytoskeleton-matrix residue remaining after detergent extraction of the cell contained the larger species of HSPG in addition to the smaller HSPG. The presence of the smaller hydrophobic HSPG in the detergent-treated cytoskeleton-matrix preparations suggests that it may form part of a transmembrane cytoskeleton-matrix linkage.

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake ( Merluccius capensis) and tilapia ( Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp 7, Leu 8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp 7, Leu 8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp 7, Leu 8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln 8-GnRH) and Trp 7, Leu 8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln 8-GnRH and Trp 7, Leu 8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp 7,Leu 8-GnRH, while tilapia brain contains both Trp 7,Leu 8-GnRH and Gln 8-GnRH. Additional GnRH molecular forms were detected in both species. The presence of multiple GnRH-related molecules in teleost fish brain suggests that the GnRH molecule has experienced gene duplication during evolution and the different structures co-opted for as yet undetermined physiological functions.

Isolation and Characterization of Four Forms of Trehalase from Rabbit Kidney Cortex

Four forms of renal trehalase were isolated and purified to homogeneity. Hydrophobic interaction chromatography separated two forms; A-form and B-form. Both forms were subdivided further on Con A-Sepharose and were stained with periodic acid-Schiff reagent, indicating that they are glycoproteins. The four forms of renal trehalase showed no significant difference in Km values for trehalose and K1 values for various inhibitors. The optimum pH of the four forms was pH 6.0 in phosphate buffer. Apparent molecular weights on gel filtration of the four forms were the same, 175,000. Furthermore, the four forms showed the same antigenicity on double immunodiffusion. However, isoelectric point (pI), susceptibility to HgCl2, stability at -80 degrees C and Na+ activation behavior were different. Glycoprotein forms were more susceptible to HgCl2 and showed lower Na+ activation than nonglycoprotein forms. The pI of less hydrophobic forms (A1, A2) was more acidic than that of more hydrophobic forms (B1, B2). On the basis of these results, it is likely that four forms of renal trehalase are "isozymes."

Characterization of two ectocellularly oriented 67 K presynaptic proteins. Lack of effect of their removal on acetylcholine release

A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1–8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase. Nevertheless, using the C1–8 monoclonal antibody, it was possible to show that they are different proteins. The C1–8 binding protein appears to be specific for the SPM in Torpedo electric organ since it was not detected in plasma membranes from the electroplaque, the electric nerve trunks or the electric lobe. The hydrophobic AChE and the C1–8 binding protein appear therefore to be useful markers of the SPM. Pronase treatment of intact synaptosomes removes most of the ectocellularly exposed proteins of the SPM, which amount to 35% of the SPM protein. Presynaptic AChE and the C1–8 binding protein are detached. But ACh release can still be induced by depolarization of the Pronase treated synaptosomes. This demonstrates that the two 67 kdalton presynaptic proteins are not directly involved in the release of the neurotransmitter.

Polymorphism of acetylcholinesterase in adult Pieris brassicae heads. Evidence for detergent-insensitive and triton X-100-interacting forms

Acetylcholinesterase (AChE, EC 3117) was extracted from Pieris brassicae heads, by successive homogenizations yielding low-salt soluble (LSS), detergent soluble (DS) and high-salt soluble (HSS) fractions. In all three extracts, two distinct AChE forms were observed, sedimenting at 7.3 and 6.5 S in the presence of Triton X-100. They accounted respectively for 20–40 and 60–80% of the total AChE activity. The 7.3 S component does not interact with Triton X-100 nor with sodium deoxycholate as indicated by sedimentation and electrophoretic migrations. Thus 7.3 S form is therefore considered as a hydrophilic component. The 6.5 S form binds detergent micelles and it aggregates in the absence of Triton X-100. Its electrophoretic mobility is increased in the presence of deoxycholate. This 6.5 S form is therefore a hydrophobic species. These two components have similar substrate and inhibitor specificities and probably correspond to different cellular locations of a single enzyme species. In contrast with vertebrate hydrophobic forms of AChE, the hydrophobic variant of Pieris AChE is not converted into the hydrophilic component by mild pronase treatment. We did not observe any additional form of AChE solubilized at high ionic strength, even in the presence of EDTA. Thus Pieris heads do not apparently contain collagen-tailed AChE molecules, similar to the asymmetric forms described for vertebrate AChE.

The electric organ of Discopyge tschudii: its innervated face and the biology of acetylcholinesterase.

An ultrastructural, histochemical, and biochemical study of the electric organ of the South American Torpedinid ray, Discopyge tschudii, was carried out. Fine structural cytochemical localization of acetylcholinesterase (AChE) indicated that most of the esterase was associated with the basal lamina. Electron microscopy indicated no marked differences in the electrocyte ultrastructure between Discopyge and Torpedo californica. Discopyge electric organ possessed three molecular forms, two asymmetric forms (16 S and 13 S) and one globular hydrophobic form (6.5 S). The asymmetric 16 S AChE form was solubilized by heparin, a sulfated glycosaminoglycan, suggesting that heparin-like macromolecules are involved in the binding of the enzyme to the basal lamina. Our results show that cell-free translated AChE peptides, synthesized using Discopyge electric organ poly(A+) RNA, correspond to a main band of 62,000 daltons which probably represents the catalytic subunit of the asymmetric AChE.

Separation of hydrophobic and hydrophilic forms of γ-glutamyltransferase from human serum by hydrophobic chromatography on phenyl-Sepharose CL-4B: studies on normal sera and sera of patients with liver disease

A new method is described for the separation of hydrophobic and hydrophilic forms of γ-glutamyltransferase (GGT) present in human serum using hydrophobic chromatography on phenyl-Sepharose CL-4B columns. The analysis of sera of 30 normal healthy people showed that 71.49 ± 8.73% (mean ± SD) of the GGT was in the hydrophilic form. In sera from 52 patients with various liver diseases the major part of the total enzyme was hydrophobic GGT, contributing 67.38 ± 13.8% of the total GGT activity. Elevated total serum GGT and a hydrophobic to hydrophilic ratio of more than one helped in diagnosing hepatobiliary disease in 51 of 52 patients. Papain digestion of serum converted hydrophobic to hydrophilic GGT, which was similar to soluble form of liver GGT. The results are discussed in relation to the proposed mechanisms of release of GGT from the liver to the circulation.

Rat intestinal maltase--glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit.

Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase-glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS)--polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500,000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120,000 to 15,0000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130,000 and 145,000 daltons. The 145,000 dalton protein was absent in p-maltase and was replaced by a faint band of 140,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of a membrane-bound acetylcholinesterase form in a preparation of nerve endings from Torpedo marmorata electric organ.

We adapted a method, originally described by Israel et al. (1976) for the preparation of cholinergic nerve endings from Torpedo, to deal with a larger quantity of electric tissue. We followed the distribution of acetylcholine (ACh), ATP, acetylcholine receptor (AChR), choline acetyltransferase (ChAT), ouabain-resistant and -sensitive ATPase, lactate dehydrogenase (LDH), and acetylcholinesterase (AChE), and obtained a nerve ending fraction, without detectable contamination by postsynaptic components. This preparation consisted of closed structures of 1-5 micrometers diameter, containing synaptic vesicles. It had the capacity to synthetize and release ACh. This preparation is therefore quite suitable for biochemical analysis of presynaptic elements. We particularly investigated its content of AChE: it consists exclusively of the 6S dimeric, hydrophobic form of the enzyme. This enzyme is enriched in the nerve ending preparation, by a factor higher than that obtained for ChAT. The yields obtained for the two enzymes suggest that the hydrophobic 6S AChE form may be mostly presynaptic in Torpedo electric organs. We characterized this form as a membrane-bound, externally active enzyme in the nerve ending preparation. It may thus participate in the hydrolysis of extracellularly liberated AChE, and its abundance suggests that presynaptic AChE could play an essential role in cholinergic transmission in Torpedo electric organs and perhaps also in other cholinergic synapses.

Membrane-bound penicillinases in Gram-positive bacteria.

The penicillinases of Bacillus licheniformis, Bacillus cereus, and Staphylococcus aureus are related in structure and cellular localization to one another more closely than they are to penicillinases from Gram-negative organisms. In the latter, penicillinases are almost exclusively found in the periplasm, while the Gram-positive bacteria retain a substantial proportion as hydrophobic membrane-bound forms. We recently showed (Nielsen, J. B. K., Caulfield, M. P., and Lampen, J. O. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3511-3515) that B. licheniformis membrane attachment was achieved through a glyceride thioether modification identical to that in several Gram-negative outer membrane proteins. We now report that the membrane penicillinases of S. aureus and B. cereus also possess the modification. We do this by demonstrating isotopic labeling of these forms of [3H]palmitate, by showing that they exhibit the same response to the antibiotic globomycin which appears to inhibit processing steps specifically involving the glyceride thioether in Escherichia coli outer membrane proteins, and lastly by isolating glyceryl cysteine sulfone, the oxidation product of the modified cysteine residue. By comparing the modification-susceptible signal sequences of Gram-positive penicillinases and of Gram-negative outer membrane proteins with those of nonmodified Gram-negative penicillinases, we describe in increased detail the structural features within the signal sequence that allow modification and cleavage resulting in membrane anchorage.

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Abstract: In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (∼ 15%) of collagen‐tailed forms (mostly A12 and A8), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m‐NaCl, 5 mm‐EDTA, 1% Triton X‐100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G4 and the monomeric G1 forms, with a smaller proportion of the dimeric G2 form. (3) The tetrameric enzyme exists as a minor soluble component (GS4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G1 form presents a marked hydrophobic character, as indicated by its interaction with Triton X‐100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent‐free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris‐HCl buffer produce a larger overall yield of G1 form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G1 form apparently derives in part from a pool of GH4 form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G4) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex‐tracts, in a manner that depends on the ionic conditions. In Tris‐HCl buffer, G1 is converted into a well‐defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm‐Ca2‐ to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X‐100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.

Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl 2 but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.

Lipoprotein nature of Bacillus licheniformis membrane penicillinase.

Membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis bears a striking resemblance to the major outer membrane lipoprotein of Escherichia coli. It can be specifically labeled in vivo with [3H]glycerol, [35S]cysteine, or [3H]palmitate but not by [32P]orthophosphate. The labeled residues are located at or near the NH2 terminus of the membrane penicillinase because they can be completely removed by trypsin which cleaves a hydrophobic peptide(s) from the NH2 terminus, thereby rendering the enzyme hydrophilic. The membrane penicillinase produced by the 749/C gene carried in E. coli on phage lambda is similar to the enzyme formed in strain 749/C itself. The peptide antibiotic globomycin, which prevents processing of the E. coli prolipoprotein, severely inhibited the attachment of [3H]palmitate or [3H]glycerol to the 749/C enzyme (either in B. licheniformis 749/C or in E. coli), blocked the accumulation of penicillinase in the plasma membrane, and enhanced the formation of exoenzyme. Under the same conditions, globomycin does not prevent the attachment of palmitate or glycerol to the E. coli prolipoprotein but inhibits processing of the modified precursor to the mature lipoprotein. These results are in contrast with the lack of effect of globomycin on the RTEM-beta-lactamase of E. coli which has no detectable hydrophobic membrane form and was not labeled with palmitate or glycerol.

Enzymes in preparative organic synthesis: Coincidence of the pH optimum for catalyst effectiveness with the pH optimum for the catalyzed reaction equilibrium

AbstractThe study concerned the pH profile of the apparent equilibrium constant for synthesis of N‐benzoyl‐L‐phenylalanine ethyl ester from the respective acid and ethanol in the biphasic system chloroform + 5% (v/v) water. The substitution of water (as a reaction medium) for the biphasic aqueous–organic system shifted the pH profile toward neutral pH values. As a result the pH range thermodynamically conducive to synthesis of the final product in the biphasic system coincided with the pH optimum of the catalytic activity of the enzyme used (α‐chymotrypsin). This approach should, in principle, be considered as general: first, per se it is independent of a catalyst (enzyme) nature; second, the biphasic method helps the shift ionic equilibria involving not only organic acids, but also bases. A physical mechanism of the ionic equilibrium shift is the same is both cases, namely, a preferable extraction from water into an organic phase of one generally nonionic (more hydrophobic) form of the reagent.

Hydrophobic interaction of soluble 3':5' cyclic nucleotide phosphodiesterase with octyl-sepharose.

Soluble cyclic nucleotide 3′:5′ monophosphate phosphodiesterase (PDE) (EC 3.1.4.17) obtained from beef adrenal cortex as the 100,000 g/1.5 h supernatant is usually regarded as a very hydrophilic protein. However, when subjected to hydrophobic chromatography on Octyl-Sepharose CL 413 it reveals strong hydrophobic interaction with the column matrix. The chromatographic procedure leads to multiple but distinct forms of PDE which degrade cAMP beyond 5′AMP to inosine, via adenosine. The same metabolic pathway was previously observed with a membrane bound multienzyme sequence. Even the soluble PDE forms separated by gel chromatography (Sephadex G 200, Sepharose S 200 and Sepharose 6B) and soluble PDE of other tissue (heart) displayed the same metabolic pattern. These findings indicate a linkage between PDE, nucleotidase and deaminase activities. The intimate association of the enzyme is additionally supported by the phenomenon of “kinetic advantage” clearly observed with the most hydrophobic PDE form. Its end product, inosine, is formed more rapidly from CAMP than from the intermediate 5′AMP. This paradoxical phenomenon is explained by close physical proximity between the enzymes involved in the metabolic pathway. Furthermore, when the most hydrophobic PDE form was immobilized on Octyl-Sepharose, rather than loss of catalytic activity even higher enzyme activities were measured. It is suggested that the so-called multiple forms of soluble PDE-at least in part-represent more or less preserved forms of a native, membrane bound, multienzyme sequence which degrades cyclic nucleotides.

Silicalite-2, a silica analogue of the aluminosilicate zeolite ZSM-11

THE structure of silicalite, a new polymorph of silica which was first prepared by Flanigen et al.1, is so similar to that of the silica-rich aluminosilicate zeolite ZSM-5 (ref. 2) that we suggest that it should be regarded as the aluminium-free end member of this zeolite series. Therefore it should be possible to prepare silica analogues of other silica-rich aluminosilicate zeolites such as ZSM-11 (ref. 3), ZSM-12 (ref. 4), ZSM-21 (ref. 5) and ZSM-34 (ref. 6). We report here the preparation of what we believe is the analogue of ZSM-11, which we call silicalite-2, adopting the terminology silicalite-1 for the form prepared by Flanigen et al.1 The silicalite-1, ZSM-5, ZSM-11 and, by inference, silicalite-2 frameworks contain 4-, 5- and 6-membered rings linked to form a system of channels with 10-membered ring openings1–3. In silicalite-1 and ZSM-5 the channels are a combination of linear and zig-zag1,2, while in ZSM-113 and silicalite-2 all the channels are linear. The lack of substitutional aluminium results in silicalites having no significant catalytic or cation exchange properties compared with the zeolites. However, silicalites are unusual in that they are the only known hydrophobic forms of silica and are capable of absorbing organic molecules up to ∼6 A kinetic diameter. Thermogravimetric studies of silicalites-1 and -2 containing absorbed long straight-chain hydrocarbon compounds (C4–C16) show that degassing occurs in two stages, which we consider to be due to self-blocking of the channels at intersection points.

Pectin Enzymes

Background: Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries.Results: The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 Å resolution) and pH 8.5 (1.93 Å resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel β helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182–187. These observed differences are due to the different pH values of crystallization.Conclusions: The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein–carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.

A new concept in preparation techniques of powders

The problem was to prepare a free-flowing, readily dispersable powder of CN (“tear gas”, an oily, sticky solid that is a respiratory irritant, having a low melting point) in the 1–10 μm size range. Past powder preparation techniques had been unsatisfactory due primarily to agglomeration. A modification of a new technique used a silica aerogel that had been chemically converted to a hydrophobic form (containing a bonded silicone compound) to coat the small particles of CN. This modification did not alter the chemical properties of the irritant material. The coating affords protection for the CN from moisture and gives excellent storage characteristics. The resultant powder was free flowing and readily dispersable from a low energy blower.