Clupeine fractionation

Table VII-1. Comparison of clupeine fractions obtained by different authors in their early studies... 

Fig. VII-5. Analytical rechromatography of clupeine fractions Y, ZI and ZII using buffered columns, resulting in chromatograms a, b and c, respectively. Sulfate specimens (Y, ZI and ZII fractions) obtained by preparative chromatography using buffered columns (see Fig. VIM) were eluted from buffered alumina with 0.48 M K2HPO4, pH 9.0— 9.2 at 25 °C (from Ando, T., and Sawada, F., 1961)...

Fig. VII-6. Countercurrent distribution of clupeine fractions Y (a) and ZII (b) obtained by preparative chromatography using a buffered column (Fig. VII-4). Solvent system ...

The fractionation of partial hydrolyzates of clupein by adsorption chromatography on filtrol-neutrol (Waldschmidt-Leitz and Turba, 1940, 1941) and on the cation exchange resin Wofatite C (Rauen and Felix, 1948) have been studied. Considerable fractionations could be obtained, but no definite peptides were identified. 

It is apparent that there are many procedures for preparing whole protamines. With typical monoprotamines such as clupeine and salmine from mature testes or sperm, a combination of the procedures described in Sections A. 2 and C. 2. c) oc or C. 2. c) P will give a high yield suitable for routine use. Although it may serve as a pure specimen of protamine, it usually consists of a mixture of several components. For special purposes which require the use of homogeneous components, such a specimen must be subjected to further fractionation (see Chap. VII). 

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). 

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). 

Fractionation of Whole Clupeine into Y and Z Fractions by Column Chromatography on Buffered Alumina (Homogeneous Clupeine Z)... 

Elution chromatography on alumina, introduced by Scanes and Tozer (1956) for the fractionation of clupeine, was slightly modified to give better resolution by eluting the protein from a buffered alumina column (Ando and Sawada, 1960). 

The specimen of clupeine sulfate (200.9 mg) was adsorbed on a column (1.88 x 46 cm) of alumina buffered with 0.48 M phosphate buffer, pH 9.0—9.2, and eluted with the same buffer at 25 °C at the flow rate of 14 ml/20 min/tube. Two main peaks, Y and 2, were obtained. The latter was tentatively divided into two parts, 21 and 2II as shown in Fig. VII-4. Each of these three fractions gave a single peak at the expected position after rechromatography on a buffered alumina column as shown in Fig. VII-5 a, b, c (Ando and Sawada, 1961). 

Desalting and Recovery of Clupeine Y and Z Hydrochlorides from the Chromatographic Fractions... 

Each of the combined fractions Y and Z obtained from clupeine sulfate (513 mg) was neutralized to pH 6—7 by adding 1 N HCl. Each solution was adsorbed on a small column (1.5 X 3—6 cm) of Amberlite CG-50 (T5rpe II) which had been buffered at pH 7 with 0.48 M... 

Since the reproducibility of chromatography using alumina is not always satisfactory, chromatographic systems using various ion exchangers were examined for the fractionation of whole clupeine. 

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. 

Fractionation of Trinitrophenylated (TNP-) Whole Clupeine into TNP-YI, TNP-Z and Free YII Components by Column Chromatography on CM-Cellulose (Homogeneous TNP-Clupeine YI, TNP-Clupeine Z... 

Clear separation of the clupeine Y fraction into two components, YI and YII, is now desired. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) is known to selectively modify amino groups in a peptide chain to yield trinitrophenyl (TNP) derivatives but never to react with imino groups (Okuyama and Satake, 1960). Thus, the N-terminal alanine residue of clupeine YI and Z should be trinitrophenylated while the N-terminal proline of clupeine YII is not, so that modification of whole clupeine with TNBS will give TNP-clupeine YI, TNP-clupeine Z and free clupeine YII. Some differences are then expected in the power of a resin to adsorb free and trinitrophenylated protamine components. 

Fractionation of Whole Clupeine into Its Three Components, YI, YII and Z, and of Whole Salmine and Iridine into Some of Their Components by One-Step Elution Chromatography on a Column of CM-Sephadex or Bio-Gel CM (Homogeneous Clupeine YI, YII and Z Homogeneous Salmine AI, Iridine I and II)... 

Unmodified whole clupeine from Pacific herring has now been fractionated successfully on a preparative scale by one-step elution from a column of CM-Sephadex C-25 or Bio-Gel CM-2 into its three components, clupeine YI, YII and Z as shown in Fig. VII-8(A) (Ando and Watanabe, 1969). The homogeneity of each compo-... 

Fig. VII-7. The chromatographic fractionation of TNP-ated clupeine into TNP-clupeine YI, TNP-clupeine Z and free clupeine YII. TNP-ated whole clupeine (8.1 mg) was added on a column of CM-cellulose (0.9 x 89 cm). It was developed with 0.1 M acetate buffer, pH 3.51, containing 0.6 M sodium chloride. The flow rate was 2.3 ml per fraction per 10 min at room temperature. The column was covered with aluminium foil to protect the TNP-derivatives from photodecomposition. The effluent from the column was analyzed by Sakaguchi colorimetry (ODsoo mix) and spectrophotometry at 340 mfi.-OD500 m x Sakaguchi color value.

Four different batches of clupeine specimens from Clupea pallasii were separated by means of this chromatographic system. The fractions of the peaks of YI, YII and Z obtained were 34 6%, 36 5% and 26 4%, respectively. The amount of F, a minor fraction that was eluted before the YII fraction (cf. Figs. VII-7, 8), was 4 3%. Thus about 96% of whole clupeine is composed of the three main components, YI, YII and Z, and their average ratio in whole clupeine is 1 1 0.7s [Suzuki and Ando, 1968 (2)]. 

Whole clupeine from Clupea harengus was also fractionated by chromatography into three components, clupeine YT, YTI and Z (see Chap. VIII. E) (Chang, Nakahara and Ando, to be published), just like clupeine from Clupea pallasii. 

Fig. VII-8. Column chromatographic fractionation on preparative scale of protamines into all or some of their components using CM-Sephadex C-25 or Bio-Gel CM-2. A. Protamine ca. 25 mg of whole clupeine sulfate (from Clupea pallasii) in a small amount of water. Column 0.9 X 130 cm of Bio-Gel CM-2 (high capacity). Elution 0.05 M acetate buffer, pH 5.8, containing 1.5 M NaCl at room temperature at a flow rate of 2.4 ml/h. B. Protamine ca. 50 mg of whole salmine sulfate (from Oncorhynchus ketd) in a small amount of water. Column 0.9 X 150 cm of Bio-Gel CM-2 (high cap.). Elution 0.05 M acetate buffer, pH 5.8, containing 1.0 M and 1.5 M NaCl at room temperature at a flow rate of 10 ml/h. C Protamine ca. 600 mg of whole iridine sulfate (from Salmo irideus) in as small as possible amount of the eluting buffer containing 0.5 M NaCl. Column 3.0 x 146 cm of CM-Sephadex C-25. Elution 0.05 M acetate buffer, pH 5.8, containing 1.5 M NaCl at room temperature at a flow rate of 85 ml/b (Reproduced from Ando, T., and Watanabe, S., 1969)...

Whole thynnine sulfate prepared from the isolated sperm cell nuclei of tunny (Thymus thymus) (Waldschmidt-Leitz and Gutermann, 1966) was fractionated into four fractions, W, X, Y, and 2 (Bretzel, 1967), by chromatography on buffered alumina according to the method used for clupeine and iridine. These four fractions were not homogeneous, although the Z fraction was at first thought to be so. 

Fig. VIII-2. Chromatographic fractionation and identification of acetyl oligopeptides obtained by selective chemical degradation products from clupeine Z (2.2 fxmoles). Amberlite CG-50 column (0.95 x 30 cm), flow rate of 5.5 ml/h, 30 °C. Recovered color value, 90% (tube No. 1—250) (from Ando et al. 1962 Iwai et aL, 1971)...

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