From clupein

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... 

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)...

Nakahara, C., Iwai, K., Ando, T. Fractionation of arginine-containing peptides obtained from clupeine Z component (in Japanese). Seikagaku (J. Japan. Biochem. Soc.) 39, 463—472 (1967). 

In 1899 Kossel isolated a similar substance from the protamine, clupeine, of herring milt, and since then E. Fischer and his pupils have obtained it from caseinogen, horn and other proteins. The preparation from horn, when racemised, corresponded in properties with the synthetical a-aminoisovalerianic acid. 

AOT/isooctane Protamines from sperm head nucleoproteins of fish Conformational studies on model polypeptides with amino acid sequences similar to clupeine and salmine were carried out in RMs [286]... 

Protamines. Strongly basic proteins of low molecular weight (4,000 to 8,000). They are rich in arginine. Examples are clupein from herring and scombrin from mackerel. 

Usually isolated as the sulfate B-2HjSO white powder, [u — 85.49" (said aq soln). One gram dissolves in 80 ml water at room temp. Freely sol in hot water, separates from the supersatd soln on cooling as a clear, colorless oil contg 50% HjO. n 1.4435. Clupeine is split by protaminase, ac-... 

Since two-thirds of the residues in clupein are arginine it follows that the sequence Arg.Arg must occur frequently in the molecule, and the dipeptide has often been isolated. Felix and Schuberth (1942) have described a method for the preparation of Arg.Arg and obtained 12.5 g. of the pure diflavianate from 50 g. clupein. 

The flat nonselective absorption observed by McLaren for clupein may be ascribed with some confidence to light-scattering, in view of its obvious extension to long wavelengths outside the limits of the protein absorption band proper, though confirmation of this explanation is clearly desirable. For a less concentrated solution of very pure clupein it was observed in this laboratory (Beaven et ed., 1950) that the absorption from ca. 3300 A. down to the onset of the short ultraviolet hands was practically negligible and flat. 

Ordinates electrophoretic velocity of Si02 particles suspended in the 0.06% sol, expressed in arbitrarily chosen units. Since it follows from experimental observed coating curves at both pH — 2 and pH = 11 that the clupein film on SiOg particles is already complete from 0.006% clupein onwards, the chosen sol concentration signifies an at least 10 fold margin of safety. 

From this we may conclude, that also quantitatively a charged colloid (here Na arabinate) behaves towards an oppositely charged one (here clupein) in the same way as a large organic ion (here germanin), but with the simplification that the systematic difference between reciprocal anion number and equivalent weight is much smaller (or perhaps absent) if the anion in question is a colloid anion. 

In this connection the following data taken from Kossel are of importance for us clupein sulphate is readily soluble in hot water but separates out from the... 

Crick, and Vand for the form factor for helical structures requires that the orders of Bessel functions for the successive layer lines from 0 to 8 be 0, 3, 6, 9, 12, 9, 6, 3, and 0. The layer-line intensities agree satisfactorily with this prediction, in the region from layer line 4 to layer line 8. There is an unexplained blackening near the meridian for layer lines 2 to 4, which, however, differs in nature for sodium thymonucleate and clupein thymo-nucleate, and which probably is to be attributed to material between the polynucleotide chains. 

Sperm heads are treated with dilute mineral acid (hydrochloric or sulfuric) (Kossel, 1929). By adding a solution of anion such as picrate to the acid extract, protamine is precipitated as an insoluble salt, which is further converted into sulfate or hydrochloride. In the original method of preparation of a protamine from testes of Rhine salmon, Miescher (1897) used 1—2 % hydrochloric acid for the extraction and platinum chloride for the precipitation of protamine. Kossel (1929) used 1 % sulfuric acid while Stedman and Stedman (1951) used 0.1—0.5 N sulfuric acid in their complete extraction procedure . Block et al. (1949) isolated the sulfate salt of clupeine and salmine on a larger scale, using 0.2 N hydrochloric acid and metaphosphate, respectively, as the extracting and precipitating reagents. 

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). 

---