Cuttlefish sperm protamines

Abstract

The sequence of very basic proteins such as protamines (more than 50% arginines) and related peptides has been determined using mass spectrometry in conjunction with Edman degradation. The capabilities of three mass spectrometric (MS) techniques [fast-atom-bombardment (FAB), ' "Cf plasma desorption ("'CFPD) and electrospray (ES)] have been evaluated on stallion protamine 1, cuttlefish protamine, and the corresponding cleavage peptides. In contrast to FAB-MS and "' Cf PD-MS, ES-MS made possible an easy determination of the molecular mass of the intact protamines ( NN 8 kDa). With ES-MS about 0.2 nmol was sufficient to yield a mass measurement with an accuracy of 0.05%. On peptides smaller than 3500 Da, both FAB-MS and ' "Cf PD-MS allowed mass measurements with an accuracy of 0.1%. 252Cf PD-MS appeared more sensitive than FAB-MS by ~ about a factor of 10. FAB-MS is nevertheless particularly interesting since in most cases it produced spectra with intense A-type fragmentation ions which provided reliable primary structure information. Protamines are highly basic proteins, associated with DNA in sperm nuclei. They are characterized by a very high arginine content (more than 50%). Purifications of protamines and cleavage peptides from enzymic digestions by ionexchange chromatography or reverse-phase HPLC are difficult because of close similarities in the amino acid composition. Primary structure elucidation of such proteins is also particularly difficult when using conventional methods of protein analysis. On purified protamines and cleavage peptides, amino acid analysis yields poor information because of the disproportion between the arginines and the other residues, especially hydrophobic residues. Moreover, serine and threonine, which often appear in the amino acid composition of such polypeptides, are partially destroyed during acid hydrolysis and cannot constitute a reference for the quantification. Primary structure elucidation of such polypeptides by automated Edman degradation is often ambiguous because the repetitive yield of the phenylthiohydantoin of arginine decreases rapidly when arginines are consecutive. New methods for purity evaluation, as well as for sequence determination, would therefore be very useful in the field of protamines.

Zinc-Induced Conformational Transitions of Acidic Peptides: Characterization by Circular Dichroism and Electrospray Mass Spectrometry

“…These shapes can be explained by the fact that seven and eightcharges, respectively, are the most populated ionization states on account of the electrostatic repulsive effects present between the charges of two adjacent side chains, as was observed for the tripeptide (Arg) 3 and for protamines which appear to be only half-protonated in the positive-ion mode. [48,49] The addition of 0.4 equiv Zn 2 /Asp has practically no effect on the shorter peptide (Leu-Asp-Asp-Leu) 3 -Asp (Table 2 and Figure 8 A), as already observed by circular dichroism. The longer peptides, (Leu-Asp-Asp-Leu) 6 -Asp (Table 2 and Figure 8 B) and (Leu-Asp-Asp-Leu) 8 -Asp (Table 2 and Figure 9 B), bind Zn 2 ions.…”

Transition of nuclear proteins and chromatin structure in spermiogenesis of Sepia officinalis

“…The most important previous studies about spermiogenic composition and protein changes in cuttlefish come from the group of Dr. Sautiére (Couppez et al, 1987;Rousseaux-Prévost et al, 1988;Martin-Ponthieu et al, 1991;Schindler et al, 1991;Wouters-Tyrou et al, 1991;Martin-Ponthieu et al, 1994). These works put together demonstrate that in the spermiogenic nuclei of Sepia the histones are substituted by two microheterogenous forms of the molecule called T, which turns out to be the precursor protamine (Fig.…”

Squid spermiogenesis: molecular characterization of testis-specific pro-protamines

“…The molecular masses of the variants TI and T2 of the squid testis-specific protein T, of the protamine Sp2 and of some peptides were determined by electrospray MS (ES-MS) either on a VG BIO-Q quadrupole mass spectrometer as described in [10] or on a VG Biotech BioQ mass spectrometer (VG Biotech, Altrincham, U.K.) with a mass range of 4000 Da. Scanning was usually performed from m/z = 400 to m/z = 1400 in 10 s with the resolution adjusted so that the m/z = 998 peak from horse heart myoglobin was 1.5-2.0 Da wide at the base.…”