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We think it worthwhile to other experimenters to mention the following observations. Recently it was noted that gel chromatographic separations of the five fragments resulting from the degradation of cytochrome c by bromocyanogen became dependent on the batch number of the gel. This dependency concerns only separations of unprotected fragments, using 7% (v/v) formic acid as the eluent. The elution profile of protected compounds, which in turn run in 50% (v/v) formic acid, was unaffected.

The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively. Journal of Endocrinology (1994) 141, 153–162

Current anti-HIV-1 screening tests combine an excellent anti-HIV-1 sensitivity with a sensitivity of only 28–93% for anti-HIV-2 positive plasma or serum samples. The reactivity of anti-HIV-2 sera in anti-HIV-1 screening tests is based mainly on the immunological cross-reactivity of the GAG and POL proteins of HIV-1 and HIV-2. We describe here a sandwich immunoassay, in which HIV-1 viral lysate is combined with an HIV-2 ENV synthetic peptide, corresponding to the immunodominant envelope epitope, as the coating antigens on microELISA plates. This immunoassay has a sensitivity of 100% for anti-HIV-1 (128 sera tested) and 100% for anti-HIV-2 (109 sera tested). Assay specificity with fresh human donor sera was 99.9% (2256 sera tested).

To study the putative binding sites of the neurotrophic peptide Org 2766, an analogue of ACTH(4–9) [H-Met(O2)-Glu-His-Phe- d -Lys-Phe-OH], biotinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4–10 μM of biotinyl-Org 2766 (b-Org 2766). Binding of both N - and C- terminally biotinylated Org 2766 was seen to phase-bright, round cells with thin processes, but not to flat, orthogonal-shaped cells with tapering processes. The b-Org 2766 binding was displaceable by an excess of nonbiotinylated Org 2766. Light and electron microscopy showed that the biotinylated peptide binds to a cytoplasmatic component as well as to the cell membrane. Double-labeling experiments with b-Org 2766 and an antibody (RT-97) to a high molecular weight neurofilament protein in dorsal root ganglion cultures showed, using fluorescence and confocal scanning laser microscopy, that all b-Org 2766 binding cells were neurofilament positive. Biotinylated Org 2766 did also bind to the neuronally differentiated cells in cultures of the human neuroblastoma cell line SK-N-SH, but not to those differentiated into epithelial cells. The present data suggest that the neurotrophic peptide Org 2766 binds specifically to cell types with neuronal characteristics.

The hydrolysis of peptide-amides in the solid state at 4 degrees due to the presence of residual strong acid was investigated. It was found that even small molar excesses of these acids cause substantial deamidation in one day. The degree of amide hydrolysis depends not only upon the pKa and the volatility of the acid, but also upon the accessibility of the amide function; peptides with bulky C-terminal residues are more stable than the less-hindered ones.

The N-terminal pentacosapeptide of Cowpea Chlorotic Mottle Virus (CCMV) was synthesized as the Nα25-acetyl, Cα25-methylamide. The compound contains six arginyl residues, which were incorporated in the peptide chain protected by protonation. The compound was obtained from an azide and a carbodiimide mediated condensation, unifying three fragments. The identity of the compound was proved by amino acid analysis, 13C-n.m.r. and 1H-n.m.r. spectroscopy. It behaved like the native protein in its tendency to interact with viral RNA (fragments).

Horse heart cytochrome c was cleaved with cyanogen bromide. The largest fragment, [Hse65]cytochrome c-(1-65)-pentahexacontapeptide lactone, was used in condensations involving four analogues of the complementary cytochrome c-(66-104)-nonatriacontapeptide. Two of the latter compounds were obtained from a semisynthesis starting with a partially protected fragment N epsilon 86-88,99,100-penta(methylsulfonylethyloxycarbonyl)cytochrome c-(81-104)-tetracosapeptide (also arising from a cyanogen bromide-mediated degradation) and analogues of the middle part, cytochrome c-(66-80)-pentadecapeptide, which were prepared by organochemical synthesis. Two other analogues of the cytochrome c-(66-104)-nonatriacontapeptide were prepared entirely by organochemical synthesis. Each of the covalently recombined analogous cytochromes c could retain an electron in the presence of oxygen and transfer it to cytochrome c oxidase, although with different reaction rates and Michaelis constants. Their redox potentials varied over a broad range. The exchanges Tyr67----Phe(F) and Thr78----Val gave rise to analogues with a lower redox potential than native cytochrome c, while the exchange Phe82----Leu or Tyr97----Leu led to analogues with the same and a higher redox potential, respectively.