Heterogeneity in Protamines

Histones and protamines are present in animal cell nuclei as basic nuclear proteins associated with deoxyribonucleic acid. They are known to be inhomogeneous and probably consist of definite amounts of several components which are very similar in their nature and structure. The significance of the heterogeneity of these basic nuclear proteins has not yet been completely elucidated, though some particular activity in controlling DNA-dependent biosynthesis is sometimes attributed to each component of the histones. 

It was well known even before Kossel s time that clupeine is not homogeneous (Goto, 1902 Miyake, 1927 Kossel and Schenck, 1928). The heterogeneity of protamine was revealed in earlier studies by tentative fractionation based on the differential solubility of presumptive components, although the fractionation was quite incomplete. In later studies protamines were shown to be inhomogeneous by physical methods, and in more recent years they have been fractionated into all or some of their homogeneous components by the use of various chromatographic procedures. 

The countercurrent distribution technique showed protamines, including clupeine, salmine, iridine and mugiline to be inhomogeneous [Rauen et al.y 1953 Felix ef al, 1957 Rasmussen, 1963 Ando et aL, 1957 (1, 3) Scanes and Tozer, 1956 Ando and Sawada, 1961 Morisawa, 1957 Ikoma, 1954 (2)]. In this case, protamine esters, i.e, the methyl ester hydrochloride of clupeine (Rauen et aL, 1953) and of mugiline (Morisawa, 1957), were separated into three and two fractions, respectively, while unesterified protamine sulfates were hardly fractionated by the technique, though patterns of a heterogeneous nature were obtained. 

Before 1960, all structural studies of protamines had been performed with wholly unfractionated or only partially fractionated specimens. The C-terminal residues of most of the protamines studied were occupied exclusively by arginine and the N-terminal residues mainly by proline, while the evidence for the presence of both proline and alanine in whole clupeine obtained from Pacific herring [Ando et al. 1955, 1957 (1, 2), 1958 (2)], indicated the presence of more than two molecular species in clupeine. 

Japanese biochemists fractionated clupeine prepared from Clupea pallasii into two fractions, Y and Z, by elution chromatography on a column of alumina which had been buffered with the eluting solvent (0.45 M or 0.48 M K2HPO4, pH 9.0—9.2) (Ando and Sawada, 1959, 1961). On subsequent application of countercurrent distribution (solvent system -butanol vs, 0.12 M sodium -toluenesulfonate containing 0.40 M NaCl), the Y fraction was further separated into YI and YII, while the Z fraction was found to be homogeneous (see Chap. VII. B). 

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The problem remains to be solved, whether such heterogeneity in protamines can be ascribed to different stages of maturation of fish testes, differences between individuals of the same species, or different preparation procedures. 

Sawada, F., Ando, T. Studies on the origin of heterogeneity in protamines by means of chromatographic method. Presented at 31st General Meeting of Japan. Biochem. Soc. (Sapporo, July, 1958). Abstr. in Seikagaku (J. Japan. Biochem. Soc.) 30, 808 (1958). Scanes, F. S., Tozer, B. T. Fractionation of basic proteins and polypeptides. Clupeine and Salmine. Biochem. J. 63, 565—576 (1956). 

I The first and simplest of them are those of sperm-head nucjeo-protein [17]. Here the nucleic acid, DNA, seems to determine the structure. In the gap already mentioned between the coils of the double spiral there is no room for any more nucleotide, but only for the smaller protamine chain, as Wilkins has shown. No similar structure has been found for natural RNA but it does occur for the combination of two different artificial poly-adenosine ribose phosphate and guanine ribose phosphate. This fact, established by Rich [18], is the best example of heterogeneous mutual coiling in vitro of polymer molecules. 

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. 

The protamines are strongly basic proteins, usually electro-phoretically heterogeneous, of comparatively low molecular weight (about 6000), and composed to the extent of nearly 60% of arginine residues. They are typical proteins of the nuclei of the sperm cells of fish and certain other animals, but are usually not found in somatic cells. Protamines contain no tyrosine, tryptophan, cysteine, or methionine. 

Since the 1940s and 1950s, with the recognition of the heterogeneous nature of protamines (see Chap. VII) and with the introduction of new materials and techniques in protein chemistry, such as ionexchange, countercurrent distribution and gel filtration, various improvements have been made to the classical procedures, permitting the preparation of protamines in a milder way and without fractionation. 

On the basis of his experimental results, Felix postulated that all components of clupeine (/.., several molecular species present as a mixture in a heterogeneous protamine like clupeine see Chap. VII) have the same N-terminal sequence of 6—8 amino-acid residues and that the differences among the components must occur along the chains towards the C-terminals. He also stated that tri- or tetrapeptide sequences of monoamino acids may occur in a chain of components. Such a chain should contain a hexa- or octapeptide of arginine in order to maintain the ratio of arginine residues to monoamino acid residues at 2 1. 

Thus the heterogeneity of clupeine and iridine obtained from individual fish was confirmed, and it was concluded that heterogeneity is an intrinsic property of protamines. Its possible biological significance in the basic nuclear proteins, protamine and histone, seems to be of interest and importance but remains to be clarified (see Chap. X. C. 1). 

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