J. Biochem. 111, 157-161 (1992) Primary Structures of Sardaines Z1 and Z2, Protamines Isolated from St riped Bonito (Sarda orientalis) Yoshiko Okamoto, * Katsutoshi Kuno , * * Terushige Motohiro, * * Norio Nishi, * * * Eiko Muta, * and Shoshi Ota* *Department of Biochemist **Laboratory of F ry, Daiichi College of Pharmaceutical Sciences, Minami-ku, Fukuoka, Fukuoka 815; K ood Quality Control and Analysis, Faculty of Fisheries , Kagoshima University, Kagoshima, H agoshima 890; and -Department of Polymer Science, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, okkaido 060 Received for publication, October 8, 1991 Striped bonito protamine, sardaine, was isolated from the sperm of striped bonito (Sarda orientalis) by extraction with sulfuric acid followed by ion-exchange chromatography. The preparation gave a single band upon polyacrylamide gel electrophoresis. Sardaine consists of 34 amino acid residues, and its sequence is: Pro-Arg-Arg-Arg-Arg-Arg Ser(Ala)-Ser-Arg-Pro-Val-Arg-Arg-Arg-Arg-Arg-Tyr-Arg-Arg-Ser-Thr-Ala-Ala .-Arg-Arg-Arg-Arg Arg-Val-Val-Arg-Arg-Arg-Arg. At position 7, serine (sardaine Z1) is partially replaced by alanine (sardaine Z2). The ion spray mass spectrum shows that sardaines Z1 and Z2 have molecular masses of 4,612.49 and 4,596.09 Da, respectively. The sequence of sardaine Z1 is 100% identical with that of thynnine Z2 from tuna fish (both fish belong to Scombridae, Perciformes). Protamines are low-molecular-weight, highly basic pro boxypeptidase A (CPA) [EC 3.4.12.1], and DFP-treated teins which are found in the spermatic cells of fish (1-3), carboxypeptidase B (CPB) [EC 3.4.12.3] were obtained birds (4-6), and mammals (7-19). The newly synthesized from Sigma Chemical, St. Louis, U.S.A. protamines are phosphorylated on serine residues. The Purification of Sardaine-The extraction was conducted phosphorylated protamines are transferred into chromatin, according to Honma (23). The proteins were extracted where they replace the histones and bind to DNA to form from minced and homogenized milts by stirring in 5% nucleoprotamine. During the maturation of the sperm, the sulfuric acid for 2 h followed by centrifugation. The extrac protamine is dephosphorylated and the nucleoprotamine tion procedures were carried out repeatedly. Two volumes becomes more compact (20-22). In this compact structure, of ethanol were added to the collected extract, and the transcription is entirely inhibited and DNA is protected whole was allowed to stand overnight at O'C. The precipi from enzymatic hydrolysis. Our knowledge of the mode of tate was obtained by centrifugation at 4,000 x g for 20 min, packing and organization of DNA is still fragmentary, and and was dissolved in hot water. Insoluble materials were so, to gain a better understanding of protamine-DNA removed by centrifugation at 15,000 x g for 20 min, then interaction, it is valuable to identify the sequences of the supernatant was allowed to stand overnight once again various protamines. In the present studies, sardaine iso at O'C. The precipitate was redissolved in a small volume of lated from the milts of striped bonito (Sarda orientalis) hot water and lyophilized (Extract-I). The pH of Extract-I was purified as a single component by ion-exchange chro dissolved in distilled water was adjusted to pH 10.5. The matography, and the amino acid sequence and the molecu solution was allowed to stand overnight at 0'C, and then was lar mass were determined. The sequence of sardaine was centrifuged at 30,000 x g for 20 min, to remove the precipi then compared with the sequences of tuna fish and For tate. Proteins were precipitated by adding 2 volumes of mosan grey mullet protamines. ethanol and a few drops of diluted sulfuric acid and allowed to stand overnight at O'C. The precipitate was dissolved in a small volume of distilled water and lyophilized (Extract MATERIALSAND METHODS II). Starting Materials-Striped bonito were collected from Extract-II (200 mg) was dissolved in 0.1 M sodium the waters around Yakushima and Tanegashima islands, in acetate buffer (pH 6.0) containing 1.6 M NaCI and then was the southern part of Japan from May to June, 1985. The applied to a CM-Sephadex C-25 (Pharmacia Fine Chemi milts were removed from the freshly caught fish and then cals) column (2.5 x 80 cm) which had been equilibrated frozen immediately at 20'C. with the same buffer. Protamine was eluted by the same Reagents-All chemicals were of the best grade commer buffer. Protamine concentration of the fraction was deter cially available. Pepsin [EC 3.4.23.1], DFP-treated car mined by measuring either the absorbance at 230 nm, or the absorbance at 500 nm after the Sakaguchi reaction. The protamine-containing fractions were pooled and desalted Abbreviations: DFP, diisopropylfluorophosphate; CPA, carboxypep on columns of CM-Sephadex C-25 (3 x 15 cm) and Amber tidase A; CPB, carboxypeptidase B; PTH, phenylthiohydantoin; lite IRA-47 (2 x 25 cm). Once again the protamine DTT, dithiothreitol; FAB-MS, fast atom bombardment mass spec trometry; IS-MS, ion spray mass spectrometry. containing fractions were pooled and adjusted to pH 5.0 Vol. 111, No. 2, 1992 157 158 Y. Okamoto et al. with 0.5 N sulfuric acid to form protamine sulfate, and then liquid chromatograph (28, 29). lyophilized (CM-I). The sample (500 pmol) was dissolved in water (100 CM-I (100 mg) was dissolved in 0.1 M sodium acetate pmol/pl), and was separated by HPLC on a Develosil buffer (pH 6.0) containing 1.0 M NaCI and then was applied 3000DS-5 column (1.5 X 150 mm, Nomura Chemicals). to a CM-Sephadex C-25 column (2.5 X 80 cm) which had The elution was performed with a 0-50% linear gradient of been equilibrated with the same buffer. After washing of acetonitrile in 0.05% trifluoroacetic acid for 25 min, at a the column with 100 ml of the same buffer, elution was flow rate of 0.1 ml/min. One-sixteenth of the eluate was performed with a linear gradient (2 x 500 ml) of 1.0-2.5 M passed to the ion source. The ion spray ion source was NaCl in 0.1 M sodium acetate buffer (pH 6.0). After operated at 4,500 V with 35 psi nebulizing gas (compressed desalting, protamine was lyophilized as the sulfate (sar air) at a flow rate of 0.8 liter/min, and the interface plate daine). and the orifice voltages were adjusted at 650 and 90 V, Pepsin Digestion of Sardaine-Sardaine (18.5 mg) dis respectively. During the on-line chromatographic analysis, solved in 0.1 M sodium acetate buffer (pH 4.0) containing the mass spectrometer was scanned from m/z=380 to 800 2.0 M NaCI was digested with pepsin at 37TC (substrate: or m/z=400 to 2,000. Mass calibration was performed enzyme= 200: 1, w/w) (24). After incubation for 2 h, the using charged ions from 0.1 mM polypropyleneglycol 1,000 reaction was terminated by keeping the preparation at and 2,000 dissolved in acetonitrile/1 mM ammonium 100°C for 3 min. The preparation was diluted with distilled acetate solution (4: 1, by volume). The molecular masses water, and then applied to a CM-Sephadex C-25 column of the peptides were calculated using the automated (2.5 x 80 cm). Fractionation and desalting were carried out Hypermass computer program, which is based on the according to the procedures described above. following equation: Gel Electrophoresis-Sardaine and the digestion prod ucts were analyzed according to Reisfeld et al. (25). The gel was polymerized in a glass dish (0.75 x 7 x 8 cm), using 20 ml of small-pore solution and 8 ml of large-pore solution. Then 2 g of the sample was dissolved in 2 ,u1 of 30% sucrose and was charged on each groove. A constant current where m, and m2 are mass-to-charge ratios of any adjacent of 28 mA/slab was applied to the anode in the upper peaks, n is the charge state, H is the mass of a proton, and chamber for 1.25 h at room temperature using a Mini M is the molecular weight. protean II (Bio-Rad). The gel was stained with 0.114% Coomassie Brilliant Blue R-250 in 5% formic acid and 2.7% RESULTS methanol for 40 min, and then destained with 3.5% formal dehyde in 24.8% methanol. Purification-The extraction of protein with 5% sulfuric Amino Acid Analysis-Samples were hydrolyzed in acid from striped bonito milts yielded protamine in an vacuo at 110°C for 24h in 6 N HCI. All analyses were almost pure state. Protamine was eluted from a CM performed according to the methods of Spackman et al. Sephadex C-25 column by isocratic elution as a single (26) on a JEOL JLC-200A amino acid analyzer. component. When herring, salmon and tuna fish protamines Automated Sequence Analysis-Automated Edman deg (30, 31) were chromatographed under similar conditions, radation of proteins (2.0 mg) was performed in a JEOL they were each resolved into several components. The JAS-570K sequence analyzer, using a peptide program. chromatography of the protamine of striped bonito on Prior to sequencing, the program was run for two cycles CM-Sephadex C-25 with linear gradient elution resulted in with 2 mg of polybrene. A program with double coupling a single peak, as in the case of pike protamine (32) (Fig. 1). and double cleavage was used on the first cycle and for the proline residue.