Clupeine composition

Preparation and amino acid composition of salmin and clupein. Proc. Soc. Exptl. Biol. Med., 70, 494 (1949). With D. Bolling, H. Gershon, and H. A. Sober. 

While the physical characterization of these proteins is still very poor, their chemical composition is well known. Analyses carried out on herring protamine (clupein), on sturgeon protamine (sturine), and on protamines... 

Fish sperm contain nucleoprotamines. Upon treatment with sulfuric acid, the nucleoprotamines are reduced to nucleic acid and protamine sulfate. The chemically heterogeneous protamines of molar mass 2000-8000 thus obtained contain only a few different kinds of amino acid residues per molecule. They are relatively rich in basic amino acids, as the composition of the protamines clupeine and salmine shows (Table 29-6), and never contain cystine, aspartic acid, or tryptophan. The basic amino acids are responsible for the bonding of the protein to the nucleic acid component. 

In spite of some differences in the species of fish used and the methods employed, the fractions designated i, ii, and iii in Table VII-1 correspond to one another as regards their amino-acid content. However, there are differences in the N-terminal residues. Fraction iii, which has the simplest amino acid composition with N-terminal alanine, was the first to be purified and have its complete amino-acid sequence elucidated (Ando et aL, 1962). New methods for the fractionation of clupeine have recently been developed and are described below (this chapter. Section B). 

Fraction 2 (the second peak) contained only a homogeneous component, clupeine 2, as shown by analysis of the amino-acid composition and the N-terminus (100 % alanine), while fraction Y (the first peak) was a mixture. On further fractionation of fraction Y on a CM-cellulose column using several buffer systems over a wide pH range, two fractions were obtained Yl-rich (80—90% N-terminal alanine contaminated with 10— 20% N-terminal proline) and Yll-rich (80—90 % N-terminal proline mixed with 10— 20 % of N-terminal alanine). Complete separation of the components YI and YII was unsuccessful by this procedure. 

TNP-YI, TNP-Z and free YII were indeed successfully separated by one-step elution of modified whole clupeine from a CM-cellulose column as shown in Fig. VII-7 [Ando and Suzuki, 1966 Suzuki and Ando, 1968 (2)]. The amino-acid composition of TNP-clupeine Z agrees with that estimated for homogeneous clupeine Z (Ando et al. 1962 Azegami et aL 1970). TNP-alanine is known to yield free alanine during acid hydrolysis (Okuyama and Satake, 1960). The molar ratios of the amino-acid content of free clupeine YII and TNP-clupeine YI proved to be simple integral numbers in the structural studies (see Chap. VIII. B and C), in which accurate analysis of the amino-acid composition and the N-terminus confirmed their homogeneity. 

CM-Sephadex C-25 as used earlier for clupeine, salmine, and iridine (Ando and Watanabe, 1969). The homogeneity of each fraction, after rechromatography, was confirmed by analysis of the amino-acid composition and the N-terminus, and by disc electrophoresis. 

Table VIII-1. Amino-acid composition of clupeine Z (Amino-acid N as a percentage of...

Peaks obtained by column chromatographic separation (Fig. VIII-1) of tiyptic peptides in a 20-h digest of clupeine Z were analyzed for their amino-acid composition, amino termini, etc. to determine the structure and amounts of peptides involved... 

A rechromatographed specimen of clupeine YII obtained unmodified by the chromatographic fractionation of TNP-(trinitrophenylated) whole clupeine (cf. Chap. VII. B. 3), has the molecular formula Arg2o Pro3 Ala2 2 Val2. This is based on the analysis of amino-acid composition and a molecular weight of about 5,000 for the hydrochloride as estimated by the DNP method (MW calculated from the amino acid composition 4,047 as a free base or 4,777 as the hydrochloride). 

Clupeine obtained from sperm cell nuclei of Clupea harengus has been studied for about 50 years, since the time of Kossel, by European protein researchers, and facts have accumulated on some features of the chemical structure of unfractionated (whole) clupeine. Over 10 years ago, some differences were noted in the amino-acid composition and the N-terminal amino acid residue of clupeine specimens prepared from Norwegian Sea (Felix et al.y 1950 Waldschmidt-Leitz et aLy 1951) and Pacific [Ando et al.y 1952,1953,1957 (1,2)] herring. It was considered by both the German and the Japanese workers that such differences were due to the species specificity of the materials used. 

In 1963, Felix and Hashimoto revised their earlier results on the N-terminals and the amino-acid composition of unfractionated clupeine from Clupea harengus so that there was no longer any essential difference in material from the two herring species. A sample of unfractionated clupeine from North Sea herring sperm heads was prepared again in the late Prof. Felix s laboratory in Frankfurt am Main in March 1963 and sent to Prof. Ando s laboratory in Tokyo for use in structural studies. This sample was found to be not different from unfractionated clupeine from Pacific herring as regards amino-acid composition, N-terminal amino acids (proline, 36% and alanine, 64%), N-terminal sequences (Pro-Arg and Ala Arg-Arg ), and tryptic peptides (Thr.Thr 0.95, Thr Arg 0.84, Val Ser Arg 1.7, Pro Val Arg 0.33, Pro He Arg 0.12, Ala Gly Arg 1.03, Ser Arg 0.52, Ala Ser Arg 0.27, Ala Arg 3.54, Ser-Ser-Ser-Arg-Pro-He-Arg 0.7g, Pro-Arg O.87, Ala Ser Arg Pro-Val-Arg I.O2, Arg 8.16, Arg-Pro-Arg 1.5, Ala-Arg-Arg 0.5, Arg-Arg 10.2 moles/3 moles clupeine) (Nukushina, 1964 Nukushina 1964). 

Ando, T., Ishii, S., Yamasaki, M., Iwai, K., Hashimoto, C., Sawada, F. (3) Studies on protamines. I. Amino acid composition and homogeneity of clupeine, salmine and iridine. J. Biochem. (Tokyo) 44, 275—288 (1957). 

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