Single Amino Group Research Articles

Cell Growth-Inhibiting Properties of Selected Carrier-Bound, Monoamine-Coordinated Platinum(II) Compounds

In contradistinction to the ligand arrangement in coordination compounds of the square-planar cis-diamminedichloroplatinum(II) type as represented by the prototype anticancer drug cisplatin, scant attention has been paid in cancer research to those platinum complexes in which only a single amino group (in addition to different ligands) is coordinated to the metal. In continuation of earlier research in this laboratory focused on macromolecular compounds containing monoamine-coordinated platinum, we have now synthesized a series of such polymeric platinum conjugates and assessed their antiproliferative activity in cell culture tests conducted against several human cancer cell lines. Conjugates containing hydroxylated side groups as hydrosolubilizing entities are found to exhibit poor, or no, activity up to the highest drug concentration tested (50 μg Pt/mL). In contrast, those conjugates in which the solubilizing units are characterized by the presence of potentially cationic tert-amine side chain terminals show remarkably high cell-killing activity, with IC50 in the range of 1–6 μg Pt/mL against the HeLa cervical epitheloid carcinoma line. Two selected samples tested against the A-2780 human ovarian cancer line and its cisplatin-resistant A-2780-cis counterpart shows lack of cross-resistance with cisplatin (resistance factor ≤1). These findings augur well for the development of polymer-conjugated monoamine-coordinated platinum compounds with carcinostatic properties.

Enantioseparation of d- and l-amino acids by a coupled system consisting of an ion-exchange column and a chiral column and determination of d-aspartic acid and d-glutamic acid in soy products

A coupled system, consisting of a conventional ion-exchange column and a chiral column, was used for separation of the d- and l-enantiomers as constituents of an amino acid mixture. The ion-exchange column was connected to an amino acid analyser and the eluents necessary for the chiral column were delivered by an HPLC pump. With this on-line system the d- and l-forms of eight of the proteinogenic amino acids were separated. An alternative method was also investigated, in which single amino acids or groups of amino acids were fed to the chiral column via a three-way divider, in order to subject the column to less aggressive conditions. By using aspartic acid and/or glutamic acid as an index of racemization and, if necessary, increasing the number of peaks investigated, a simple and fast method for the determination of the degree of racemization was achieved. The method was applied to three soy products. The products contain 3.4–5.8% d-aspartic acid and 1.4–2.4% d-glutamic acid

Geometric nomenclature and classification of RNA base pairs.

Non-Watson-Crick base pairs mediate specific interactions responsible for RNA-RNA self-assembly and RNA-protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2'-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson-Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.

Study on single-bond interaction between amino-terminated organosilane self-assembled monolayers by atomic force microscopy

Chemically modified atomic force microscope (AFM) tips and modified silicon wafers with self-assembled monolayers (SAMs) were produced using 3-aminopropyltriethoxysilane (APTES) molecules. Prior to modification, the silicon wafers were thoroughly cleaned by improved RCA method, producing hydrophilic and smooth surfaces. The bare and modified silicon substrates were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). In this paper, a statistical analysis method, which makes use of the properties of the Poisson distribution, was applied to investigate the interaction between the tips and silicon surfaces covered with SAM terminating in amino groups. The single-bond force between a single amino group pair in water was obtained from the total adhesion forces measured by AFM and was found to be 200.0±43.6 pN. This value was assigned to the result of mainly hydrogen bond interaction between a single NH 2–NH 2 pair. Comparisons between our results and those measured by means of Johnson–Kendall–Roberts (JKR) theory and other methods were also made.

Reaction with, and fine structural recognition of polyamines by human IgE antibodies

Human IgE antibodies from nine allergic subjects were found to react with poly- L-lysine (PLL) and other polyamines. Radioimmunoassay inhibition studies indicated that the two amino groups, but not the carboxyl, in lysine contributed to the antibody binding and 4-aminomethyl-1,8-octanediamine, a compound containing three primary amino groups, was a better inhibitor than compounds containing only two primary amino groups. Ethylamine showed weak but clear inhibition indicating that even a single amino group could bind to the antibody combining site. Substituted ethanolamine and quaternary ammonium compounds were well recognized by some sera but with others, substitution hampered recognition. Inhibition studies with compounds containing an amino and a carboxyl group at different distances revealed that an adjacent carboxyl group interfered with recognition of the amino group by some IgE antibodies. IgE binding to PLL was examined at different pHs and ionic strengths. Binding was greatest at pH 5–6 to 8 and decreased markedly outside this range. Ionic strengths higher than 0.3 M significantly diminished the binding. These results indicated that binding of specific antibody to polyamine was due to electrostatic interactions of positively charged amino groups in the polyamine with the antibody combining site. These results may be relevant to mechanisms underlying recognition of some allergens in some atopic conditions.

Preliminary toxicological studies of selected water-soluble polymer-platinum conjugates

The objective of this preliminary investigation of a number of water-soluble carrier-bound platinum(II) complexes for potential use in cancer chemotherapy was to assess the toxicological behavior of representative platinum coordination compounds anchored to, or incorporated into, polymeric carriers via polymer-attached amine ligands. The conjugates included linear polyaspartamides (1-4, 6, 7), each composed of a major fraction of subunits featuring side-chain-attached tertiary amino groups as water-solubilizing entities, and a minor fraction of subunits comprising the anchored platinum complexes, again as side-chain components. Whereas in 1-4 the platinum atom was polymer-bound through a single amino group, both 6 and 7 contained polymer-attached cis-diamine-chelating ligands coordinating to the metal center. Also included in this study was a linear polyamidoamine (5), which contained a poly(ethylene oxide) segment in the backbone in addition to intrachain ethylenediamine segments acting as cis-diamine chelating ligands for coordination to the platinum center. The compounds were injected as aqueous (phosphate-buffered saline) solutions into the tail veins of CD-1 mice (four to eight mice per conjugate), and the maximally tolerated dose was determined for each compound. For polyaspartamides 1-4 the dose levels ranged from about 25 mg Pt (kg body weight -1 ) (in conjugate 4) to 500 mg Pt kg -1 (in compound 1), the latter conjugate proving some 100-fold less toxic than cisplatin (3-4 mg Pt kg -1 ), which was included in this study for comparison. Low toxicity (tolerated dose 160 mg Pt kg -1 ) was also observed for the intrachain cis-diamineplatinum complex polymer (5). The polyaspartamide conjugates 6 and 7, on the other hand, both characterized by a cis-diamineplatinum complex system in the side chain, were toxic even below the dose level of 20-25 mg Pt kg -1 . The preliminary findings of this study, while providing a basis for more extensive and broad-based toxicological studies, will serve to direct and optimize structural conjugate designs in forthcoming synthetic programs.

Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system?

To investigate systematically the effect of polyamidoamine (PAMAM) dendrimer size, charge, and concentration on uptake and transport across the adult rat intestine in vitro using the everted rat intestinal sac system. Cationic PAMAM dendrimers (generations 3 and 4) and anionic PAMAM dendrimers (generations 2.5, 3.5, and 5.5) that were modified to include on average a single pendant amino group were radioiodinated using the Bolton and Hunter Reagent. 125I-Labelled dendrimers were incubated with everted sacs in vitro and the transfer of radioactivity into the tissue and serosal fluid was followed with time. The serosal transfer rates seen for all anionic generations were extremely high with Endocytic Indices (EI) in the range 3.4-4.4 microL/mg protein/h. The concentration-dependence of serosal transfer was linear over the dendrimer concentration range 10-100 microg/mL. For 125I-labelled generation 5.5 the rate of tissue uptake was higher (EI = 2.48+/-0.51 microL/mg protein/h) than seen for 125I-labelled generations 2.5 and 3.5 (0.6-0.7 microL/mg protein/h) (p < 0.05). The 125I-labelled cationic PAMAM dendrimers (generations 3 and 4) displayed a tissue uptake (EI = 3.3-4.8 microL/mg protein/h) which was higher (p < 0.05) than the rate of serosal transfer (EI = 2.3-2.7 microL/mg protein/h), probably due to nonspecific adsorption of cationic dendrimer to the mucosal surface. As the anionic PAMAM dendrimers displayed serosal transfer rates that were faster than observed for other synthetic and natural macromolecules (including tomato lectin) studied in the everted sac system, these interesting nanoscale structures may have potential for further development as oral drug delivery systems.

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking.

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.

15N enrichments of casein and plasma protein amino acids in cows ingesting 15N-labelled ammonium sulphate.

The aim of this work was to determine by ion-exchange liquid chromatography and isotope ratio mass spectrometry the specific 15N enrichment of amino acids in casein and plasma proteins in cows receiving three successive daily oral doses (300, 150 and 150 g) of (15NH4)2SO4 (10 atom per cent isotopic enrichment) and to examine the 15N enrichments obtained with regard to nitrogen transport and metabolism in the lactating cow. To investigate the 15N distribution in amino acids in casein and in plasma proteins, samples of 15N-labelled casein and plasma proteins were extracted either from a pool of several milkings (36-96 h after starting to administer the tracer) or from pooled venous blood (removed on the fourth day after the start of administration) from the four lactating cows. 15N enrichments of the proteins studied, expressed as atoms percent excess, were 0.2509 for casein and 0.0577 for plasma protein. Chromatographic fractionation of the amino acid mixture (protein hydrolysates) resulted in nine groups containing between one and four amino acids: Asp, Ser and Thr; Glu; Pro; Gly; Ala; Val and Met; Ileu and Leu; Tyr; Phe; His and Lys; and Arg. High 15N incorporation was demonstrated in all individual or groups of amino acids studied. In both proteins, Glu appeared to be the most enriched amino acid, Phe and Arg the least enriched. Most aliphatic molecules with a single amino group were highly enriched. The much lower (3.5-7.7-fold) enrichments in plasma protein compared with casein suggest considerable intracellular dilution at the site of liver protein synthesis. Finally, the amino acid separation methods are discussed and suggestions for improving them considered.

Footprinting of echinomycin and actinomycin D on DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine.

In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.

Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector

We describe a stable chelating linkage for the reversible immobilization of oligohistidine tagged proteins in the flow cell of the ‘BIAcore’ surface plasmon resonance (SPR) biosensor. The carboxymethylated dextran surface of the flow cell was covalently derivatized with N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA ligand) via its single primary amino group, and the derivatized surface charged with Ni 2+. 6His-VP55, an N-terminally tagged derivative of the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was immobilized to this surface in a manner that was dependent upon the immobilized NTA ligand, the prior injection of Ni 2+ at a concentration of > 10 −5 M and the 6His tag, and which was reversible upon injection of EDTA. The stability of immobilization varied inversely with the amount of 6His-VP55 immobilized and was greatest in buffer of pH 8.0 or greater, containing NaCl at a concentration of 0.1 M. Utilizing these conditions, 6His-VP55 remained stably immobilized during 60 min of buffer flow at moderate flow rates. VP39, the stimulatory subunit of vaccinia poly(A) polymerase, interacted with the immobilized 6His-VP55. ∼99% of immobilized 6His-VP55 molecules were available for VP39 binding, in contrast to the ∼40% availability for 6His-VP55 molecules immobilized covalently, via primary amino groups. Three additional proteins, tagged at either the N- or C-terminus with oligohistidine, were shown to be stably immobilized via the chelating linkage. This simple method permits immobilization of proteins in the BIAcore biosensor via a commonly employed affinity tag, in a stable and reversible manner, and requires only a single biosensor flow cell for the iterative generation of immobilized protein surfaces.

Car☐yacyl derivatives of cardiolipin as four-tailed hydrophobic anchors for the covalent coupling of hydrophilic proteins to liposomes

Two car☐yacyl derivatives of cardiolipin, O-succinyl- and O-glutarylcardiolipin, were synthesized with the aim of using them as artificial membrane anchors for the immobilization of hydrophilic proteins to liposomes. Four adjacent fatty acid residues can be introduced into a protein with only one single amino group being blocked, by reacting the cardiolipin derivatives with the protein amino groups after carbodiimide activation. α-Chymotrypsin, used as a model protein, and modified with on average two molecules of O-succinylcardiolipin was incorporated into liposomes, which had been prepared by different methods, with very high yield. If incorporated in performed liposomes, the car☐yacyl cardiolipin anchors were also efficient in binding proteins to liposomal surfaces. Up to 350 μg chymotrypsin / μ mol lipid were coupled to small unilamellar vesicles, preserving reactivity of the enzyme towards specific macromolecular inhibitors. Human IgG could also be bound to anchor-containing liposomes with high protein to lipid coupling ratio as well as high coupling yield.

Molecular features necessary for the uptake of diamines and related compounds by the polyamine receptor of rat lung slices

The influence of 17 putrescine analogues on the uptake of putrescine and/or paraquat by rat lung slices has been determined. Most of these compounds are competitive inhibitors of putrescine and/ or paraquat uptake, but three show no inhibiting activity. Apparent K i values of the putrescine derivatives increase, and thus the inhibitory effects decrease, with increasing N-methylation. Comparison of N-methyl-1,4-diaminobutane ( K i , = 8 μM) with N, N′-bis-methyl-1,4-diaminobutane ( K i = 25.5 μM) shows that a single primary amino group is desirable for high inhibiting activity. Dimethylation at one amino function does not greatly decrease inhibitory potential (thus N, N′-dimethyl-1,4-diaminobutane has K i = 11.5 μM). Increasing the size of N-alkyl substituents in putrescine derivatives, decreased their inhibitory action on the uptake of putrescine. Investigation of the effect of conformationally-restricted analogues of putrescine shows that both ( E) and ( Z) isomers of 1,4-diaminobut-2-ene are poor inhibitors of putrescine uptake. Analogues of putrescine with bulky substituents on the butyl chain, i.e. the meso-and rac-isomers of l,1-dichloro-2,3-diaminomethylcyclopropane, do not inhibit putrescine uptake. Inhibiting putrescine derivatives which contain aziridine groups are competitive inhibitors of putrescine and paraquat uptake. Surprisingly, N-(4-aminobutyl)aziridine is the most effective inhibitor of putrescine uptake studied, and is a better inhibitor of paraquat uptake than the endogenous polyamine, putrescine. N-(4-Aminobutyl)aziridine binds reversibly to the polyamine transporter and its inhibitory effects do not appear to be due to any cytotoxic activity of the aziridine. The parameter A (mM) −1 defined as 1000 K i (where K i units are μM) was taken as a measure of the affinity of a compound for the polyamine receptor in this paper.

Do cardiotoxins possess a functional site? Structural and chemical modification studies reveal the functional site of the cardiotoxin from Naja nigricollis

Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure—activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin γ from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin γ are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adajacent loops rich in β-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin γ involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.

Chromatographic enzyme immunoassay for T-2 toxin

Both the active ester maleimide moieties of the cross-linking reagent, N-[(γ-maleimidobutyryl) oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1 : 1.1 M conjugate of anti-T-2 toxin Fab′ and RNase (Fab′-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1 μg of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab′-RNase complexed to a T-2 agarose affinity gel.

Photoinduced electron transfer within complexes between plastocyanin and ruthenium bisbipyridine dicarboxybipyridine cytochrome c derivatives

A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis

Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.

Hormonotoxins: Preparation and characterization of ovine luteinizing hormone-gelonin conjugate

Abstract With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.

Affinity separation with polyaldehyde microsphere beads

Agarose polyaldehyde microsphere beads were prepared by encapsulating polyaldehyde microspheres of various diameters, e.g., polyacrolein or polyglutaraldehyde microspheres, within agarose beads. Amino ligands such as proteins or drugs can be bound covalently to the beads in a single step at physiological pH. The binding capacity of the beads towards various amino ligands is inversely related to the diameter of the microspheres encapsulated in the agarose matrix. Different reagents, e.g., bovine serum albumin, ethanolamine and hydroxylamine, were studied as blocking reagents of the free aldehyde groups. Blocking the remaining aldehyde groups after coupling the amino ligands to the beads is essential for increasing or retaining the reactivity of the ligands conjugated to the beads. Among the reagent studied, hydroxylamine was found to be the most suitable blocking reagent of the free aldehyde groups of beads conjugated with proteins. The extent of leakage of amino ligands bound to the agarose-polyaldehyde microsphere beads was studied as a function of the pH of aqueous solutions of the beads. At physiological pH the leakage was negligible. At acid pH, leakage of ligands containing several primary amine groups, e.g., proteins, was insignificant. However, significant leakage was detected for ligands containing a single amino group. The leakage of proteins bound to the agarose-polyaldehyde microsphere beads was found to be much less than the leakage of the same proteins bound to agarose beads through the cyanogen bromide activation method.