Irwin M. Chaiken

Semisynthetic Peptides and Protein

Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.

Analytical affinity chromatography in studies of molecular recognition in biology: a review.

Measuring macromolecular and cellular interactions remains central to the study of recognition in biology and its application in biotechnology. Analytical affinity chromatography provides a versatile methodology to detect and quantitate such interactions. Both zonal and frontal elution approaches have been developed, essentially in parallel, for analytical affinity chromatography. A close quantitative relatedness of chromatographically obtained equilibrium constants and analogous constants determined fully in solution has been found for a growing number of proteins. This consistently observed correlation has formed the basis for extending theoretical treatments in order to evaluate not only monovalent molecular systems of varying types but also multivalently interacting macromolecules, including those which exhibit cooperativity. The potential to measure chemical rate constants by affinity chromatography also has been recognized, and experimental tests of the available theory are being made. As a micromethod, the quantitative use of affinity chromatography has important applicability for biochemical analysis of an increasing array of biologically active molecules being discovered and isolated but available in only relatively small amounts. Analytical affinity chromatography thus provides a means to use matrix--mobile interactant systems to study mechanisms of biomolecular interactions and therein to attain an understanding of such interactions which often is not easily achieved by solution methods alone.

Carbon-13 Fourier transform nuclear magnetic resonance studies of peptides

Abstract The carbon-13 nuclear magnetic resonance (CMR) spectra of several small peptides have been obtained at 25.1 MHz and natural abundance of 13C using the Fourier transform technique with proton noise decoupling. Chemical shift values have been studied as a function of pH. The peptide corresponding to the 1–15 sequence of ribonuclease has been synthesized both with normal and 15% 13C enriched Phe in position 8, and the CMR spectra of these two products are compared.

Active conformation of an inactive semi-synthetic ribonuclease-S.

We have studied the integrity of folded structure of a fully active semi-synthetic ribonuclease-S which lacks amino acid residues 16 through 20, and an inactive one with the same residues deleted and 4-fluoro-l-histidine substituted for active site histidine 12. Using “Y” form crystals, we obtained X-ray structural data to a resolution of 2·6 A and, incorporating phase information calculated from refined ribonuclease-S coordinates, prepared several types of electron density maps. These showed that the overall backbone structure and active site configuration of both analogues do not differ noticeably from those of the native protein. Structural homology extends to the catalytically relevant side-chain at position 12; 4-F-His† assumes the same position as does His in active ribonuclease-S. This supports the view that the 4-F-Hisl2 analogue is inactive due to a change in histidine 12 imidazole basicity, rather than to any significant conformational distortion within the active site.

Mapping and isolation of large peptide fragments from bovine neurophysins and biosynthetic neurophysin-containing species by high-performance liquid chromatography

Abstract A peptide mapping procedure suitable for rapid analysis and peptide recovery was devised for the neurophysins. Tryptic fragments of performic acid-oxidized bovine neurophysins I and II were fractionated by reverse-phase high-performance liquid chromatography using γ-cyanopropyl-bonded columns. Elutions were achieved using a gradient from triethylammonium phosphate buffer to mixtures of this buffer with increasing proportions of acetonitrile. All tryptic fragments, except for dipeptides, were separated. Assignments of eluted peaks to particular authentic neurophysin peptides were achieved by collection of peaks and determination of their amino acid compositions. The use of this peptide mapping procedure to detect subpicomole amounts of neurophysin peptides in cell-free biosynthetic products was demonstrated.

Quantitative affinity high-performance liquid chromatography of neuroendocrine polypeptides using porous and non-porous glass derivatives

Analytical affinity HPLC was developed to isolate and characterize neuroendocrine peptide/protein components. Bovine neurophysin II (NP-II) was covalently immobilized on succinamidopropyl derivatives of both controlled-pore glass (CPG) and non-porous glass (NPG). These derivatives were packed into 25 X 0.46 cm I.D. stainless-steel columns and incorporated into a high-performance liquid chromatograph. Interaction of [3H]Arg8-vasopressin ([3H]AVP) with NP-II was examined by chromatography of AVP on both CPG and NPG affinity matrices. Zonal elution profiles of [3H]AVP on NPG matrix showed, as predicted theoretically, a linear dependence of retardation on the concentration of hormone injected. The data permit calculation of the equilibrium dissociation constant for the NP-II/AVP interaction. Elution characteristics also were measured by frontal analysis of large-zone chromatography experiments, the results of which were in good agreement with the zonal elution analysis. Affinity resulting from dimerization also was studied by chromatography of [125I]NP-II on the NPG matrix. In this case, concentration dependence of retardation was non-linear, again as predicted theoretically. Off-rate kinetic constants for dissociation of the mobile interactant from the stationary phase also were obtained. The studies illustrate the utility of analytical affinity HPLC on non-porous beads for measuring relative affinities for various soluble ligands with small amounts of material. Chromatography on the CPG column proved useful for purification of microscale amounts of [3H]AVP.

Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Abstract Methionyl-tyrosyl-phenylalanyl-ω-aminohexyl-agarose was synthesized and shown to be suitable for both the affinity chromatographic purification of neurophysins and the measurement of the ligand binding parameters of these proteins by quantitative affinity chromatography. Bovine neurophysin I binds to the tripeptidyl matrix in 0.4 m ammonium acetate, pH 5.7, conditions under which no binding occurs with the parent ω-aminohexyl-agarose. Subsequent elution can be effected with 0.2 m acetic acid. The affinity matrices obtained have capacities for neurophysin of up to 4 mg/ml gel bed volume and therein provide for the convenient purification of the neurophysins by a two-step buffer-acid elution. [Carbamoyl-14C]neurophysin I also binds to the ligand-agarose matrix. Using this labeled protein, competitive elution analysis was performed by one-step elution of zones of protein with the binding buffer in the presence of varying amounts of soluble competitive ligand, lysine vasopressin. The characteristic decrease of elution volume of labeled protein with increasing soluble, competing ligand concentration indicates that the affinity matrix interacts biospecifically with neurophysin. This analysis allows the binding affinities for both soluble vasopressin and immobilized tripeptide ligand to be quantitated.

Relationship between α-helical propensity and formation of the ribonuclease-S complex

Abstract Variant semisynthetic ribonuclease-S complexes were characterized in which the helical glutamic acid 9 residue was replaced by either leucine or glycine. The Leu-9 and Gly-9 synthetic peptides, corresponding otherwise to residues 1 through 15 of bovine pancreatic ribonuclease, were studied with respect to the ability to bind, and generate enzymic activity, with the complementary native protein fragment containing residues 21 through 124 of ribonuclease (RNAase-S-(21–124)). Both the Leu and Gly peptides bind to the RNAase-S-(21–124) to yield complexes with catalytic properties similar to those obtained with the Glu-9-containing peptide of residues 1 through 20 of ribonuclease (RNAase-S-(1–20)). However, whereas the binding affinity of Leu peptide to RNAase-S-(21–124) is only a factor of three less than that for RNAase-S-(1–20), that for Gly peptide is about 20-fold less than that for RNAase-S-(1–20). The stronger binding of Leu than Gly peptide corresponds to the observed propensity of leucine but not glycine for the α-helical conformation in globular proteins. In spite of the weakened affinity of the Gly peptide for RNAase-S-(21–124), it is essentially fully as capable as the Leu-9 and RNAase-S-(1–20) peptides in directing the re-formation of correct disulfide-containing conformation of RNAase-S-(21–124) after disulfide randomization of the latter.

Interactions and uses of antisense peptides in affinity technology

Antisense peptides, amino acid sequences encoded in the antisense strand of DNA, can interact with significant affinity and selectivity with their corresponding sensepeptides. Experimentally, sense-antisense peptide recognition has been observed repeatedly. However, skepticism about the biological relevance of this phenomenon has persisted. This is due in part to the unexpected and somewhat couterintutive nature of the interaction as well as to its non-universality as an empirical observation. Nonetheless, antisense peptides in several cases investigated so far have been used as immobilized ligands for the successful affinity chromatographic separation of native (sense) peptides and proteins. For example, immobilized antisense peptides corresponding to Arg8-vasopressin (AVP) have been used to separate vasopressin from oxytocin chromatographically as well as to affinity capture AVP-receptor complex. These results, together with improved understanding of the general features of amino acid sequence which drive antisense-sense peptide interactions as well as new ideas for making antisense peptides chimeras, are beginning to suggest improved ways to make antisense-related peptides as affinity agents for separation as well as for other biotechnology applications.

Preparation of biologically active conjugates of bovine neurophysins and other polypeptides with multi-(poly-d,l-alanyl)-poly-l-lysine and their use to elicit antibodies

Abstract A new, chemically specific method was developed for the preparation of functionally and immunologically active conjugates of proteins, via amino groups, to modified multichain (poly- d,l -alanyl)-poly- l -lysine. With this method, soluble conjugates were prepared for bovine neurophysins I and II. The isolated protein-polymer conjugates displayed high levels of biological activity as judged by the non-covalent binding of specific peptide ligand, indicating that the conformation of the protein moiety is minimally perturbed in these species. Rabbits immunized with the neurophysin conjugates produced antisera containing specific antibodies as detected by Ouchterlony double-immunodiffusion assays. All sera from rabbits immunized with neurophysin conjugate precipitated with conjugate as antigen, a phenomenon not observed for sera from rabbits injected with unmodified neurophysin. The presence of neurophysin-specific antibodies in the conjugate antisera was indicated by competition assays using unmodified neurophysins. For neurophysin I conjugate antisera, this presence of neurophysin-specific antibodies was confirmed by the demonstration of binding activity for [ 14 C-carbamoyl]neurophysin I in two radioimmunoassays. The first was based on specific elution of antibody-bound antigen on Sephadex G-100; the second, on binding of antibody-antigen complex to protein A on the surface of Cowan I strain Staphylococcus aureus . Specific neurophysin I antibodies could be fractionated by affinity chromatography on neurophysin I-Sepharose. In order to demonstrate the applicability of this method to other systems, conjugates also were prepared with bovine pancreatic ribonuclease. These derivatives were enzymically active and elicited high titers of ribonuclease-specific antibodies.