The amino acid sequence of the third component of clupeine

375

PRELIMINARY NOTES

BBA 31007 The amino acid sequence of the third component of clupeine*

Clupeine from Pacific herring (Clupea !ballasii) is composed of nearly equal quantities of 3 main species of molecules, clupeine YI, YII, and Z 1. The chemical structures of the 2 species, YIV and Z 2, have already been determined. The third component, YI, was purified as its TNP-derivative 1 and submitted to structural analyses. Clupeine YI is composed of the following 31 amino acids : Al%, Pr%, Sera, Thr 2, Gly D Ile~, and Argo0 as estimated using a Hitachi KLA-3B automatic amino acid analyzer. The molecular weight based on this composition is 4839 as the hydrochloride. The sequence of 16 amino acid residues from the C-terminus of TNP-clupeine YI, determined by the alternate action of carboxypeptidases A and 13 together with hydrazinolysis, was Pro-Arg-Arg- Arg-Thr-Thr-Arg-Arg-Arg-Arg-Ala-GlyArg-Arg-Arg-Arg-OH. Elucidation of the total amino acid sequence was achieved by 2 degradative procedures, hydrolysis by trypsin and by thermolysin. The tryptic peptides of TNPclupeine YI were fractionated on an Amberlite CG-5o column and IO peptides and arginine were identified (Fig. I) using essentially the same method as previously

T & 1.5~

u

/

li

Z

~.- 1.0 '.',

~'o:~ l ::i!I:', m~

lr

At9

I I

/~

A II

5o'" 0

AIo-GIy-Ar~ .

" O0

HSer~-Arg-Pro-lle-Arg

~ ~ ~ ~

I I I I

./"

I/" ~"

250 ,. 300 Tube No.

~-~

./t ~

350

~

I

- i I

I

i < , o" I LArg-~Arg, Pro)'Arg

l 400

, 450

Fig. I. F r a c t i o n a t i o n and identification of t r y p t i c p e p t i d e s from T N P - c l u p e i n e YI. The digestion m i x t u r e of T N P - c l u p e i n e Y I (31. 5 mg) w a s applied to t h e t o p of a c o l u m n (o. 7 c m × 51 cm) of A m b e r l i t e CG-5 o T y p e e q u i l i b r a t e d w i t h the starting buffer (o.i M borate buffer, p H 8.o). The c o l u m n w a s first d e v e l o p e d w i t h t h e starting buffer. A f t e r 154 tubes h a d been collected, an e x p o n e n t i a l gradient s y s t e m w a s applied. T h e m i x i n g c h a m b e r w a s filled with 500 m l of starting buffer. To t h e reservoir, o.i M b o r a t e buffer (pH 8.35 ) c o n t a i n i n g 0. 5 M NaC1 w a s added. The c o n t e n t of the reservoir w a s replaced w i t h o.i M b o r a t e buffer (pH 8.5) c o n t a i n i n g 1. 5 M NaC1 after 284 tubes had been collected. The effluent was collected in 3.o m l p o r t i o n s w i t h a flow r a t e of 3.0 ml per t u b e per 27 rain a t 3 o°. The elution of peptides was followed b y S a k a g u c h i c o l o r i m e t r y (absorbance at 500 mtt) a n d s p e c t r o p h o t o m e t r y at 34 ° m # . The recovered color yield (Sakaguchi) was 99 % (tubes 1-59o ).

II,

A b b r e v i a t i o n : T N P - , 2,4,6-trinitrophenyl- ; D N P - , 2,4-dinitrophenyl- ; PTC-, p h e n y l t h i o carbamyl-. * P r e s e n t e d at t h e I 7 t h S y m p o s i u m on P r o t e i n Structure, t~yoto, N o v e m b e r , 1966. Biochim.

Biophys. Acta, 14o (1967) 375-377

376

PRELIMINARY NOTES

published a. F r o m the results on t r y p t i c p e p t i d e s a n d the above C-terminal sequence analyses, the following p a r t i a l s t r u c t u r e was f o r m u l a t e d for T N P - c l u p e i n e Y I : T N P Ala-Arg-(Ser3-Arg-Pro-Ile-Arg, 6Arg)-Pro-Arg3-Thr 2-Arg~-Ala-Gly-Arg4-OH. In order to d e t e r m i n e t h e t o t a l a m i n o acid sequence of clupeine YI, larger p e p t i d e f r a g m e n t s m u s t be o b t a i n e d , r e t a i n i n g bonds between arginine residues intact. N e i t h e r the selective chemical cleavage of the N ~ O acyl r e a r r a n g e d p r o d u c t nor c h y m o t r y p t i c digestion was sufficiently successful in the case of T N P - Y I . Success was achieved b y the use of a b a c t e r i a l t h e r m o s t a b l e protease. T N P - c l u p e i n e Y I was digested b y t h e r m o l y s i n 4,5 o b t a i n e d from Bacillus thermoproteolyticus R o k k o (digestion: 3.5 h in o.oi M calcium chloride, w i t h p H s t a t at p H 8.0 a n d 40°; m o l a r r a t i o of s u b s t r a t e to enzyme, 119: i). P e p t i d e s in t h e digestion m i x t u r e were f r a e t i o n a t e d b y g r a d i e n t elution column c h r o m a t o g r a p h y on CM-eellulose (Fig. 2). The s t r u c t u r e of p e p t i d e s in 5 m a i n peaks of the c h r o m a t o g r a m were established b y d e t e r m i n a t i o n of a m i n o acid composition, b y use of the D N P - a n d PTC-methods, b y stepwise c a r b o x y p e p t i d a s e A a n d B digestion a n d b y hydrazinolysis. These 5 t h e r m o l y s i n p e p t i d e s c o m p l e t e l y account for the composition of whole T N P - c l u p e i n e YI. Thermolysin was thus found to be a new excellent tool for s t u d y i n g the a m i n o acid sequence of basic proteins, since tile e n z y m e h y d r o l y z e d no a r g i n y l a r g i n i n e bonds at all.

!

0.7

~

T I ~0.5 E '

0.3

I

40.6

i, ',AIo - G l y - A r g 4 / . / 11

o

O

F

TNP - AI~ -Arg4- Set TNP-AI0- AP~4

/il

/'/[le-Arg

4

Pro-Ar93-Thr

i

4o4i

1 /"

/ :L

~ r ) - Ser2-Arg-Pr°. , ' ' /

I

I

o.2 8

o~0.1 o 2O0 Tube No.

25O

I~" ,'

3 0 0 "'

550

0

Fig. 2. Chromatographic separation and identification of peptides in a thermolysin digest of TNPclupeine YI. The digestion mixture (from 2.27 #moles of TNP-clupeine YI) was applied to a column (o.9 cm x 15o cm) of CM-cellulose. Peptides were first eluted with the starting buffer (o.i M acetate buffer, pH 6.o; tubes 1-8o), then an exponential gradient elution was applied (81-514). The mixing chamber contained IOOO ml of starting buffer and to the reservoir o.I M acetate buffer (pH 6.5) containing i.o M sodium chloride was added. The peptides remaining on the column were eluted with o.i M hydrochloric acid. The flow rate of the effluent was 3.89 ml/tube per 18 min. The effluent was analyzed by Sakaguchi colorimetry and spectrophotometry at 34° m/~. The color yield (Sakaguchi reaction) was ioi ~o (tubes 1-555). F r o m the i n f o r m a t i o n o b t a i n e d on t h e r m o l y s i n a n d t r y p s i n p e p t i d e s a n d also b y C-terminal sequence analyses, it was possible to deduce t h e complete a m i n o acid sequence of clupeine Y I which is shown below, along w i t h t h e p r e v i o u s l y established s t r u c t u r e s of t h e o t h e r c o m p o n e n t s of clupeine: Y I chain: H - A l a - A r g 4 - S e r 3 - A r ~ - P r o - I l e - A r g 4 - P r o - A r g a - T h % - A r g 4 - A l a - G l y Arg4-OH.

Biochim. Biophys. Acta, 14o (1967) 375-377

PRELIMINARY NOTES

377

YII chain: H- Pro-Arg3-Thr-Arg~-Ala-Ser-Arg-Pro-Val-Arg4-Pro-Arg~-ValSer-Arg4-Ala-Arg4-OH. Z chain : H-Ala-Arg4-Ser-Argz-Ala-Ser-Arg-Pro-Val-Arg4-Pro-Arg2-ValSer-Arg4-Ala-Arg4-OH. The primary structure of clupeine YI is somewhat different from those of the other 2 components, YII and Z, but a close inspection of the structures of these 3 molecular species reveals that their gross structures are essentially the same, even though the individual amino acid sequences are different from each other in details. Except for clupeine, none of the components of the basic proteins, protamines and histones, associated with DNA in sperm and somatic cell nuclei, has ever been separated homogeneously and completely analysed with regard to its amino acid sequence. Clupeine is composed of only 3 main components and characterization for homogeneity and primary structure of all three have now been accomplished. Thus clupeine specimens will provide excellent natural material for investigations designed to discover possible differences in the interaction 6 of basic proteins with DNA and in the biological functions of the resulting deoxyribonucleoproteins. This work was supported in part by a Scientific Research Grant from the Ministry of Education in Japan.

Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo (Japan)

TOSHIO ANDO

KOICHI SUZUKI

I T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, 121 (1966) 427 . 2 T. ANDO, K. IWAI, S. ISHII, M. AZEGAMI AND C. NAKAHARA, Biochim. Biophys. Acta, 56 (1962) 628. 3 T. ANDO, S. ISHII AND M. YAMASAKI,Biochim. Biophys. Acta, 34 (J959) 60o. 4 S. ENDO, J. Ferment. Technol., 4 ° (1962) 346. 5 H. MATSUBARA, A. SINGER, R. SASAKI AND T. H. JOKES, Biochim. Biophys. Res. Common., 21 (1965) 242. 6 S. INOUE AND T. ANDO, Biochim. Biophys. Acta, 129 (1966) 649.

Received April I3th, 1967 Biochim. Biophys. Acta, 14o (1967) 375-377

B~A 310O6

An autolyzed and still active form of bovine trypsin Bovine trypsin is a single peptide chain with an N-terminal isoleucine residue 1. However, the crystalline enzyme always contains additional N-terminal residues --valine, serine, aspartic acid (or asparagine)--which are eliminated by repeated crystallizations of the DFP-inhibited derivative s. Similarly, when a trypsin sample prepared from pure trypsinogen is submitted to chromatography s on CM-cellulose at pH 6.o in the presence of Ca 2+ (13 mM), the material under the trypsin peak, although highly active, contains the additional N-terminal residues listed above: valine (o.24 mole/mole, uncorrected value), serine, and aspartic acid (0.07 mole/mole each). Only N-terminal isoleucine is found if trypsin is inhibited by DFP prior to chromatography. Biochim. Biophys. Acta, 14o (1967) 377-38o