N-terminal sequence of horse spleen apoferritin

.\RCHIVES

OF

BIOCHEMISTRT

.\ND

N-Terminal

BIOPHYSI(‘S

113,

Sequence

of Biochemistry,

(1966)

of Horse

ASITA Depurtntent

l-4

Spleen

,4. SURAY

University of Californicr, San Fruncisco, Culifornia

Received

Apoferritin

September

San

Francisco

Mrtlicd

C’erlter,

30, 1965

4 rlillhydrill-llegative, argillitle-collt:tiIlillg peptide was isolated after tryptic drolgsis of horse spleen apoferritin. It was found to be acetylated :tlld to have struct,ure, .~-t~ce~~1seryl~srr~1~g1utarr~i~r~l~isoleuc~l-argi~~ir~e. This pelltapeptide the T~trrmi~~al sequence of the subulrit rhaill of apoferritill.

@onveut,ional luckhods for determining an S-tenuin:tl mnino wid, the fluorodinitrohetmenc method of Sanger and t,he T’,dnmn rewtion, have failed to reveal a free terminal residue for horse aplecn apoferritin (1, 2). Blthough a citatiou in n related lmlw (3) states that a&y1 groups have been detect)ed in apoferritiu, no det’ails were given. There have been no reports on sequence studies with apoferritin. In this study x method for t’he fractionation of tryptic l)eptides of apoferrit,in on Dowex 50 c~olunmz is presented. The Ntermiual pent~apcptide sequence is given and the termiual amino acid is shown to be Nac*etylserine. MATERIALS

ANI)

METHOIX

Horse spleen apoferritin was prepared by the modification of the method of Granick and Michaelis (4, 5) and digested with trypsin as described previously (5). When the try@ digest was adjusted to pH 4.2%l.6 with glacial acetic acid, a gelatinous precipitate appeared. Either this complete digest, or the supernatant fraction only was applied to the chromatographic column. Chromatographic separation of peptitles. The tryptic peptifdes from the complet,e digest were fractionated on 1.2 X 150.cm columns of l)owex 50-X2, 200-400 mesh (Biorad, Richmolld, CaliforIlia) operated at room temperature. The resin was 1 The author was supported by a USPHS Trairling Grant 5-TlGM372 and by a research grant from the Xational Science Foundat,ion GB-2080 (to H. Tarver and R. A. Fineberg).

hythe is

equilibrated with I’,; pyriditle buffer at pH 50. (a stock solution of lo’,; pyriditre was brought, to pl1 5.0 by addition of glacial acetic acid, and dilutions were made for use as specified). An empirically determilled ionic strength gradient was delivered to 1 he column from a tlille-chambered Autograd (Techrlicoll Chromatography Corp., Chauncey, New York) ; 100 ml each of the followillg cotrcentrations of the stock pyridine huffel were used: 1, 1, 1, l.i, 3.3, 3.3, 6.6, 0.6, and 107;,. The last peaks were elutcd with 500 ml of the 105, buffer applied at the end of the gradient. The pept,ide of interest ill this study was foulld to be ill the superllatallt of the acidified tryptic digest. A sharper separation of this peptide from ndjacelit peaks was obtailred on the same c*olumtl by using a lower ionic strength gradielrt composed of 100 ml each of the following concentrations of the stock pyridille buffer: 0.25, 0.25, 0.5, 0.5, and lTO. Buffer was delivered to all columns at a corlstarrt, rate of 3.0 ml per minute. The column effluent was split, three ways: Two streams were sent to the analytical system and the third was collected at the rate of one tube every 2 minutes. The ninhydrin reaction alld a Sakaguchi reaction (for peptides containing arginine) were run (JII the Autoanalyzer. Details of these reactions are shown schematically in Fig. 1. rimino acid determinati0n.s. Quantitative amino acid determinations were made using a slightl?; modified syst,em (5) with an Autoanalyzer (Technicon). Qualit,ative est,imates of amillo acid COXLtent, and tentative identifications were obtained by thidayer chromatography on 0.1.mm cellulose plates (Machery-Nagle MN300, Brinkmann Instr., Long Island, Kew York). (;ood resolution of glu-

2

SUKAN

tamine, glutamic acid, serine, and glycine was obtained by using met,hanol-pyridine-water (160:8:40) in the first direction followed by 80% phenol ill the second. !l’he hydrazinolysis reaction. The hydraxinolysis SAKAGUCHI

REACTlON

wbromosuccinimi

NINHYDRIN

REACTION

b%

%%eto

FIG. 1. Schematic representation of automated ninhydrin and Sakaguchi reactions. Reagents were: 8-hydrosyquinoline, 0.02% in 3 N NaOH; N-bromosuccinimide, 0.170 in HSO; stock ninhydrin, 20 gm of ninhydrin and 1.5 gm of hydrindant,in were dissolved in 650 ml methyl cellosolve, then 350 ml of 4 X sodium acetate (pH 5.5) was added. The solution was stored under nitrogen. Ninhydrin reagent, the stock solution was diluted with four parts of 50’7; methyl cellosolve. Nitrogen was passed over t,he solution during use.

FIG. 2. Fractionation fractionation procedure in Fig. 1.

of trypt.ic are given

method of Narita (6) was used to identify the S-acyl residue of the ninhydrin-negative peptide. Standards of acetyl hydrazide were prepared from both ethyl acetate and A-acetyl isoleucine at the same time as the reaction was run wit,h the peptide. Each of the two single dimensional chromatographic systems of the above author was used on thin-layer cellulose plates (collidine-water, 10:2, and pyridine-aniline-water, 9: 1:4). The hydrazides were st,ained with alkaline silver nitrate. Hydrolysis with carboxypeptidases. The peptide was hydrolyzed from its C-terminal end by successive uses of carboxypeptidases B and A (Worthington Biochemicals, Freehold, New Jersey). An aliquot representing about, half a micromole of peptide in 3 ml of water was reacted with 100 pg of carboxypeptidase B at pH 8.0, and the reaction was titrated with 0.005 h: NaOH and followed to completion on a pH-stat (Radiometer, Denmark). Then 63 rg of carboxypeptidase A (suspended in 10yO lithium chloride, solubilized by briefly raising the pH to 9.6 and then adjusting to pH8.0) was added and the reaction was followed to completion on the pH-stat. The enzymes were inactivated by addition of 0.1 A7 HCl to pH 3. This preparation is designated “long-term incubation.” A second aliquot of the peptide was similarly treated with 100 pg of carboxypeptidase B; then 32 pg of carboxypeptidase A was added. However, in this case, as soon as the reaction began (noted by a drop in pH) it was terminated bJ addition of acid to pH 3. This preparation is designated

peptides of complete apoferritin ill t,ext. A schematic representation

digest, ou Dowes of the analytical

50. Details of the methods is shown

N-TERMINAL

SEQUENCE

OF

HORSE

SPLEEN

TABLE AMINO

; 0.5 4 x a 0.3 uz r 0 0.1

NINHYDRIN

tS70m~J

-

SAKAIOUCHI

f505mp)

w--s

40

60

Ro

I

COMPOSITIO~V

100

3. Resolution of Peptides 4 and 5 from supernatant fraction of acidified tryptic digest on Dowex 50. The low ionic strength buffer system was used in this separation as described in the text. A schematic representation of the analytical methods is shown in Fig. 1. FIG.

FOUK

OF PEPTIDE pmoles analyzed in sample

acid

5

Serine Glutamine Isoleucine Ammonia Arginine FRACTION

ACID

Amino 4

3

APOFERRITIN

(as glutamic

Relative ratios

1 61c1 0.97 0.71 0.63 0.76

acid)

2 1 1 1 1

a This value for serine was obtained by extrapolating to zero time the amounts found after 24 and 72 hours of hydrolysis, 1.45 and 1.12 &moles, respectively. This value represents a 90gc recovery of serine in the 24-hour hydrolyzate.

Ac-ser.ser.gluNwa.ileu.arg

L‘short-term incubation.” Solutions of each of the two enzyme pairs, inactive in 0.05 IV HCl, were carried through all separation procedures as enzyme blanks. The amino acids liberated by the enzymes were absorbed on 1.2 X S-cm columns of Dowex 50-X8, 2W400 mesh in the hydrogen form. The columns were washed with 25 ml of water which was collected and saved for later analysis. The amino acids were eluted in about 50 ml of 5 l\r NHdOH, and this solution was evaporated to dryness on a rotary evaporator at 50”. Each residue was dissolved in a small amount of water, and an aliquot was chromatographed on cellulose in the methanol-pyridine:phenol system to determine whether glutamine or glutamic acid were present. Since glutamine was found, the remainder of each of these solutions was refluxed with 2 K HCl for 2 hours to convert glutamine to glutamic acid (hydrolysis was necessary since glutamine is not resolved from serine in the analytical system). Each of the enzyme digests and enzyme blank solutions was analyzed for amino acid content. The acetylated amino acid passed t,hrough the small Dowex 50 columns and was collected in the 25.ml water washes. Each solution was evaporated to dryness on the rotary evaporator at 50”, t’hen deacetylated by hydrolyzing with 6 3 HCl at 105” in a sealed tube for 16 hours. Sample and blank solutions were evaporated to dryness and taken up in 0.1 ilr HCl. The terminal amino acid was tentatively identified by thin-layer chromatography, and then positively identified and measured on the amino acid analyzer. RESULTS

Separation tryptic

peptides

AND

DISCUSSION

of peptides. The of t’he whole digest of

and isolation

I

Carboxypeptidase

B

AC-ser.ser.gluN~~.ileu

+i

Ac-ser.-ser

+

gluNnl

+

ilau

+

arg

K,‘frjzy$

FIG. 4. Action of carboxypeptidases B and A on Peptide Four. Relative amounts of free amino acids and amino acids obtained after acid hydrolysis of acetylated fragments are indicated.

apoferritin were separated on Dowex 50 as shown in Fig. 2. Nineteen significant peaks were observed. Peak 4 was ninhydrin-negative and contained arginine as demonstrated by’ a positive Sakaguchi reaction. Better resolution of peaks 4 and 5 was effected by using the supernat,ant part of the acidified tryptic digest and t,he low ionic. strength gradient

syst,em

(Fig.

3).

Later

preparations

of PepGde 4 were made exclusively by t’his method. Composition of Peptide Few. An aliquot of Peptide 4 was hydrolyzed and found to have the composition (serin?j ‘. glutamic acid, isoleucine, ammonia, argmme) (Table I). A control aliquot was carried through t’he automatic: amino acid analysis procedure

4

SURAN TABLE

EEI,E.\SE

OF A41gr~o

II

Acms

FROM

B

C.\I~BOSYPEPTIDASES

Amino

Liberated, absorbed (plllOleS)

arid

Argilline Isoleucine Glutamine glutamic Serine (2)

0.29 0.2(i 0.15

(as acid)

PEPTIDE

0

AND

FouR

BY

&

Sot absorbed, hydrolyzed acid (pmoles)

Total (pmoles)

0 Trace 0.15

0.2Q 0.26 0.30

0.54

0.54

(1 Partit,ion of amino acids on Dowex 50 after hydrolysis with carboxypeptidases B and A. Average of values obtained in two experiments. TABLE AMINO

Scrns

LIBERATED

III FROM

CARBOSYPEPTID.~SE

Amino

Serille Glutamine acid) Isoleucine

acid

(as glutamic

PEPTIDE

FOI-K

BY

AC1 pIllOk liberated after shortterm incubation

!moles liberated after longterm incubation

Trace Trace

Trace 0.16

0.15

0.32

a Peptide Four was first incubated with carboxypeptidase B and the reaction run to completion. Carboxypeptidase A was then allowed to react with each of two aliquots of the peptide solution for different lengths of time. Details are as in text,.

without being hydrolysed, and no free amino acids were detected. The failure of this peptide to react with ninhydrin reagent suggested that it was either very large or that its N-terminal amino acid was substituted. The reaction of Peptide 4 with hydrazine yielded a mixture of amino acid hydrazides and acetyl hydrazide which was identified by each of t’he two chromatographic systems (6). Sequence of Peptide Few. Carboxypeptidase B is known t’o split arginine from a Cterminal position in peptides. The remaining

amino acids may then be removed sequentially by carboxypeptidase A; in rate studies the residues most proximal to the C-terminus of the peptide should accumulate most’ rapidly during incubation with the enzyme. The liberated amino acids and any unreacted peptide should be absorbed on Dowcx 50: the K-terminal acetylat(ed arnino acid and acetylated pept,ide fragments should pss through Dhe resin. Figure 4 outlines t’he procedure and anticipat,ed results. Table II shows the result’s of duplicate long-term incubations with the carboxypept’idases. Serylserine is the acetylated or terminal peptide residue since no free serine was found. These data suggest that the peptide sequence is N-acetylseryl . seryl . glutaminyl . isoleucyl arginine. This sequence is confirmed by a comparison of the amounts of amino acids liberated during the shortand long-term incubations of Peptide 4 wit’h carboxypeptidase A (Table III). Arginine was not measured in t#his experiment. Since only isoleucine was found after short’ contact wit’h the enzyme it must be adjacent t,o arginine in the peptide. The longer incubation produced more isoleucine, some glutamine and a trace of serine. Hence glutamine is adjacent to isoleucine and the second seryl residue must, occupy t,he position between isoleucine and acetylserine. Therefore, the sequence of Pept’ide 4 is N-acetylseryl. seryl glutaminyl . isoleucyl . arginine. REFERENCES 1. HARRISON,

P. M.,

WARING,

2.

w.

A.,

SCRAN,

AND

I.

T.,

HOFK~NN, P.,

J.

(iROSS,

!lfol.

I).,

AND

MAIN-

4, 251 (1962). unpublished obserBiol.

vations. 3.

MAINWARING, BND

HSRRISON,

W. 1. P., cited P. M., J.

by HOFMANN, T., dfol. Biol. 6, 256

(1963). 4.

S., AND MICHAELIS, L., J. Biol. Chem. 147, 91 (1943). 5. SURAN, A., AND T~RvER, H., Bach. Biochem. Biophys. 111, 399 (1965). 6. NARITA, K., Biochim. Biophys. Acta 28, 184 (1958). GRANI~K,