Fractionation and Characterization of Basic Proline-Rich

J. Biochem. 91, 2067-2075 (1982)

Fractionation and Characterization of Basic Proline-Rich

Peptides of Human Parotid Saliva and the Amino Acid

Sequence of Proline-Rich Peptide P-E

Satoko ISEMURA, Eiichi SAITOH, and Kazuo SANADA

Department of Oral Biochemistry, Nippon Dental University

Niigata Faculty, Niigata, Niigata 951

Received for publication, January 7, 1982

Basic prolinerich peptides of human parotid saliva were fractionated and charac

terized. The amino acid sequence of one of the purified peptides, P-E, was deter

mined to be

The results demonstrate the repetitiveness of the partial sequence within the

molecule and the occurrence of structures common to those of other salivary poly

peptides such as P-C and Protein C.

Salivas are known to contain various polypeptides

whose proline contents are extremely high. Al

though their physiological functions are still uncer

tain (1, 2), they have been well studied because of

their abundance, unique amino acid compositions,

and diversity of molecular species.

The present authors have reported the isola

tion and the amino acid sequences of several

prolinerich peptides of human whole saliva and have discussed relationships among various pro-

line-rich proteins and peptides (3-5).

In the present study, we have fractionated and

characterized basic prolinerich peptides of human

parotid saliva and have determined the amino acid

sequence of one of the purified peptides, P-E, for

further understanding of structural features and the

multiplicity of peptides of this class.

Abbreviations: PTH, phenythiohydantoin; SDS, so

dium dodecyl sulfate; N-, amino-; C-, carboxyl-; DNS-,

1-dimethylaminonaphthalene-5-sulfonyl; CPase, car

boxypeptidase.

MATERIALS AND METHODS

Mfaterials-The following were purchased

from the sources indicated: clostripain, Sigma;

carboxypeptidase (CPase) B and L-(1-tosylamido-

2-phenyl)ethyl chloromethyl ketone-treated trypsin,

Worthington Biochemical Corp.; CPase P, Pep-

tide Institute Inc.; thin layer precoated sheets

(HPTLC Fertigplatten Kieselgel 60 F1254), Merck;

SP-Sephadex C-25 Pharmacia; polyamide layer,

Seikagaku Kogyo Co., Ltd.; reagents and solvents

for Edman degradation, Wako Pure Chemical

Industries, Ltd.

Collection of Saliva-Parotid saliva was collected in icechilled test tubes from students of about 21 years of age, utilizing double walled suction cups and sour candy stimulation.

Vol. 91, No. 6, 1982 2067

SPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNRPQCUPP-

PPGKPQGPPPQGDKSRSPR,

2068 S. ISEMURA, E. SAITOH, and K. SANADA

Preparation of Proline-Rich Peptides-Basic prolinerich peptides were prepared according to the method described previously (4). Briefly, methanol was added to the pooled parotid saliva to make the 80%, methanol, and the precipitates formed were removed by filtration. The filtrate was concentrated in vacuo and applied to a column of Bio-Gel P-6. The first peak fraction was ap-

plied to a column of DEAE cellulose (DE 32) equilibrated with 0.01 M NH4HCO3 and the break-through fraction was collected.

Amino Acid Analysis-Amino acids were analyzed with an amino acid analyzer (JLC 6AH) after hydrolysis of peptides in constant boiling HCl in sealed evaculated tubes by the method of Spackman et at. (6). Amino acid compositions in the tables are shown in molar ratios. Numbers in

parentheses are the nearest integers or numbers determined after sequence analysis. Amino acids released by CPase B or P were analyzed by the method of Benson et al. (7) in the system of lithium citrate buffer or by the method of Spackman et al. (6).

Sodium Dodecyl Sulfate (SDS) Gradient Poly

acrylamide Gel Electrophoresis-Gel electropho

resis was carried out in a linear gradient gel sys

tem of 9.7 to 20% of acrylamide and 1/36.5 to

1/20 (weight ratio to acrylamide) of N,N•Œ-methyl

enebisacrylamide, according to the modified meth

od of Jackson and Blobel (8). Gels were fixed

in 12% trichloroacetic acid and stained with Co

omassie brilliant blue G-250 (9) or 1% phospho

tungustic acid (10).

Amino (N-) Terminal Analysis-The N-termtnus of a peptide was determined using the 1-dimethylaminonaphthalene-5-sulfonyl (DNS-) method (11) or the phenylthiohydantoin (PTH) method (12).

Sequence Analysis-The DNS-Ldman method

used was that of Gray (13). In order to minimize

the loss of peptides during ethylene dichloride ex

traction, the conventional direct Edman degrada

tion method (12) was slightly modified: trifluoro

acetic acid was removed by flushing with nitrogen

gas to make a film of peptides along the wall of

the reaction tube. DNS-amino acids were identi

fied by polyamide TLC with the solvent systems of

Woods and Wang (14), and PTH derivatives of

amino acids were identified by TLC on silica gel

with solvent systems ‡U and V of Brenner et al.

(15).For the isolation of a CPase B-treated peptide,

the digest was subjected to gel filtration through a

column of Bio-Gel P-4 (0.8 x 130 cm) equilibrated

with 0.05 M acetic acid to remove salt and the

released amino acid(s), and the resulting peptide

fraction was freeze-dried.

Conditions for enzymic digestion, mild acid

hydrolysis, and hydrazinolysis are listed in Table

IS.

RESULTS

Fractionation of Proline-Rich Peptides of Par

otid Saliva-The fraction containing basic proline

rich peptides was applied to a column of SP-

Sephadex C-25 (Fig. 1). The amino acid analysis

of fractions I to ‡\ indicated the predominance

of proline (33-47%) in all fractions. Since SDS

polyacrylamide gel electrophoresis of these frac-

Fig. 1. Separation of the DE 32 breakthrough fraction on an SP-Sephadex C-25 column (0.9 x 100 cm). The column was equilibrated with sodium acetate buffer,

pH 3.6 (0.015 M Na+ concentration). A gradient elution was performed with 500 ml each of the starting

buffer and 0.5 M NaCl in the same buffer. The arrow indicates the position at which the gradient elution was started. Fractions of 8.0 ml were collected.

J. Biochem.

PROLINE-RICH PEPTIDES OF HUMAN PAROTID SALIVA 2069

Fig. 2. Further purification of fractions ‡T, ‡Y, ‡[,

and ‡\ of Fig. 1. (a) Separation of I on a column of

Bio-Gel P-6. The column (1.7 •~ 135 cm) was equi

librated and eluted with 0.1 M ammonium acetate (pH

8.0). Fractions of 5.0 ml were collected. (b) Separa

tion of ‡Y. Conditions for the chromatography are

the same as in (a). (c) Separation of VIII on a column

of Bio-Gel P-10 (1.3 x 135 cm). The column was

equilibrated and eluted with 0.05 M acetic acid. Frac

tions of 4.0 ml were collected. (d) Separation of ‡\.

Conditions of chromatography are the same as in (a).

tions revealed their heterogeneity, further purifica

tions were attempted. Gel filtration through Bio-

Gels of fractions ‡T, ‡Y, ‡[, and ‡\ (Fig. 2) yielded

electrophoretically pure peptides 1-2, ‡Y-1, ‡[-2,

and ‡\-1, respectively. Gel filtration of fraction ‡[

-1 through Bio-Gel P-4 afforded a pure pep-

tide ‡[-1. SDS gel electrophoretograms of these

five peptides are presented in Fig. 3. Further

purification of other fractions were not successful.

Amino Acid Compositions of the Purified Pep-tides-Amino acid compositions and yields of the

purified peptides are tabulated in Table I. On the basis of the amino acid compositions, electro

phoretic mobilities, and chromatographic behav-

Fig. 3. SDS gradient gel electrophoresis of purified

prolinerich peptides. Gels were stained with l%

phosphotungstic acid for prolinerich peptides and by

Coomassie brilliant blue G-250 for standard proteins.

The standard proteins used for molecular weight esti

mation were: Whale myoglobin (17,200), CNBr frag

ments of myoglobin (14,900, 6,420), horse heart cyto

chrome c (12,300) and its CNBr fragment (7,760). (1)

a mixture of prolinerich peptides obtained as the

DEAE cellulose (DE 32) breakthrough fraction of

methanol-treated parotid saliva, (2) peptide 1-2(P-B),

(3) peptide ‡[-2(P-C), (4) peptide ‡Y-1(P-D), (5)

peptide ‡\-1(P-E), (6) peptide ‡[-l(P-F).

iors, peptides 1-2 and ‡[-2 are considered to be

identical with P-B and P-C, respectively, sequences

of which were elucidated previously (3, 4). Pep-

tides ‡Y-1, ‡\-1, and ‡[-1 are newly isolated

peptides and designated P-D, P-E, and P-F, respec

tively. Glutamic acid, proline, and glycine con

stituted about 80% of total residues in P-D, P-E,

and P-F. Lysine, arginine, aspartic acid, and

serine were also contained in these peptides. Ala-

nine was detected in P-D and P-F, but not in P-E.

Vol. 91, No. 6, 1982


TABLE ‡T. Amino acid compositions of P-B, C, D, E, and F.

a On the basis of a molecular weight estimated from the previous data (3, 4). b On the basis of a molecular weight

estimated by SDS gel electrophoresis (Fig. 3) on the assumption that the method gives a higher value than the true value.

Estimation of Molecular Weights of P-D, P-E, and P-F-Estimation of molecular weight by SDS polyacrylamide gel electrophoresis of prolinerich peptides seems not to be reliable, since a higher molecular weight (7,800) was obtained for P-C, which has a molecular weight of 4,371 as calculated from its amino acid sequence data (4). Deficiency of hydrophobic amino acids may be responsible for overestimation of the molecular weight by this method (16). Minimum molecular weights of P-D, P-E, and P-F calculated from amino acid com-

positions are 5,950, 2,120, and 5,670, respectively. On the assumption that the molecular weights of these peptides are lower than the values obtained from SDS-gel electrophoresis, the molecular weight of P-F was estimated to be equal to its minimum molecular weight. In consideration of mobilities of P-D and P-E relative to that of P-F, the molecular weights of P-D and P-E were estimated to be double and triple their minimum molecular weights, respectively.

Partial Structure of P-D and P-F-The N-termini both of P-D and P-F were determined to

be serine by the DNS-method. The N-terminal

12 residues of P-D and P-F were determined by

duplicate experiments of Edman degradation to be

as follows.

Neither of these peptides released any amino acid

upon CPase B digestion.

Glutamine (0.22 mol/mol) was liberated from P-D by the CPase P treatment, suggesting carboxyl

(C-) terminal glutamine in P-D. Similar treatment of P-F yielded Pro (0.46 mol/mol), Ala (0.54), Gln (0.58), Lys (0.71), Arg (0.79), Gly (0.96), and Ser (2.13), suggesting the localization of polar amino acids near the C-terminus of P-F.

Complete Amino Acid Sequence of P-E-N-

and C-terminal sequence analysis: The N-terminus

of P-E was determined as serine by the DNS-

method. The N-terminal sequence of P-E was

determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-

Gly-Pro-Pro-Pro-Gln-Gly-Gly-Asn-Gln-Pro- Gin-

J. Biochem.

P-D: Ser-Pro-Pro-Gly-Lys-Pro-Gin-Gly-Pro-Pro-Gln-Gln-;

P-F: Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gin-.


Gly-Pro-Pro-Pro-Pro-Pro-Gly- by 25 cycles of

Edman degradation.

The C-terminus of P-E was determined to be arginine, since 0.80 mol/mol of arginine was released by CPase B digestion. CPase P digestion released the following amino acids: Gin (0.63 mol/ mol), Gly (1.25), Asp (1.00), Lys (1.19), Ser (2.06), Pro (1.06), and Arg (2.12).

In order to determine the second residue of

the C-terminus of P-E, hydrazinolysis was applied

to the peptide isolated after the CPase B treatment.

Proline was the only detectable amino acid when

analyzed on the amino acid analyzer using the

column for analysis of acidic and neutral amino

acids.

Fractionation of peptides from clostripain diges

tion: The peptides obtained from clostripain di

gestion of P-E (see Table IS) were separated on a

Bio-Gel P-4 column as shown in Fig. IS. Frac

tions 38-44 and 46-50 contained small amounts

of impurities which were removed by chromatog

raphy with SP-Sephadex C-25 (data not shown).

Fractions 75-80 and 84-90 were used without

further purification for sequence analysis. The

four peptides Clo-‡T, Clo-‡U, Clo-‡V, and Clo-‡W

were thus obtained. The amino acid compositions

and final yields of these peptides are tabulated in

Table ‡US.

Sequence of Clo-‡T: Edman degradation of

Clo-I (0.12 ƒÊmol) revealed the N-terminal 35 resi

dues. The C-terminus was determined as arginine

by CPase B digestion. CPase P released Gin (0.58

mol/mol), Gly (1.63), Asn (1.03), and Arg (1.00).

On the basis of these results and amino acid com-

position, the sequence of Clo-‡T was deduced to be

Ser-Pro-Pro- Gly-Lys-Pro -Gln-Gly-Pro-Pro-Pro-

Gln-Gly-Gly-Asn-Gln-Pro -Gln-Gly-Pro-Pro -Pro-

Pro-Pro-Gly-Lys-Pro -Gin -Gly-Pro- Pro- Pro- Gln-

Gly-Gly-Asn-Arg.

Sequence of Clo-‡U: The N-terminal 18 resi

dues of Clo-‡U were determined by Edman degra

dation. The C-terminus of Clo-‡U was determined

as lysine by CPase B digestion. Gin (0.80 mol/

mol), Gly (1.07), Asp (0.98), and Lys (0.92) were

released from Clo-‡U by CPase P. The sequence

of Clo-‡U was determined to be Pro-Gln-Gly-Pro-

Pro-Pro-Pro-Gly-Lys-Pro -Gin-Gly-Pro-Pro-Pro-

Gln-Gly-Asp-Lys.

Sequence of Clo-‡V: After one step of Ed-

man degradation, Clo-‡V was applied to an amino

acid analyzer without hydrolysis. Since arginine

was detected, the sequence of Clo-‡V was deter-

mined to be Ser-Arg.

Sequence of Clo-‡W: Edman degradation of

Clo-‡W suggested the sequence of Ser-Pro for the

N-terminal two residues. Arginine was detected

as a free amino acid after two steps of degradation.

CPase B released 0.50 mol/mol of arginine from

Clo-‡W. Consequently, the sequence of Clo-‡W

was deduced to be Ser-Pro-Arg.

Mild acid hydrolysis: To obtain the overlap

peptides of clostripain peptides, P-E was subjected

to mild acid hydrolysis in 0.03 N HCl. The hy

drolysate was fractionated on a column of Bio-Gel

P-6 as shown in Fig. 2S. The amino acid com-

positions of H-I and H-2 are presented in Table

2S. The amino acid composition, N- and C-ter

minal analysis of peptides from fractions 29-35

suggested that uncleaved P-E was the major con

stituent of these fractions. The elution from this

column of H-1 earlier than P-E, which has a higher

molecular weight, is probably due to the charge

effect, since the former has lost two arginine and

one lysine residues (Table ‡US).

Partial sequence of H-l: The amino terminus

of H-1 was determined as serine by the DNS-

method. The N-terminal 18 residues were deter-

mined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-

Pro-Pro-Pro-Gln-Gly-Gly-Asn-Gln-Pro-Gln- by

Edman degradation. Hydrazinolysis of H-1 yield-

ed aspartic acid and glycine in a molar ratio of

0.44 : 1.00. Thus, H-I is the N-terminal fragment

with the heterogenous C-terminus.

Partial sequence of H-2: The DNS-Edman method was applied to the sequence analysis of this peptide containing 2 mol of serine, since the direct Edman method had difficulty in positioning the serine residue. The amino terminal 4 residues were determined to be Lys-Ser-X-Ser-. CPase B released 0.68 mol/mol of arginine. These data and the amino acid composition indicate that H-2 has the sequence of either Lys-Ser-Arg-Ser-Pro-Arg or Lys-Ser-Pro-Ser-Arg-Arg.

Positioning of clostripain peptides of P-E:

Since the N-terminal 25 residues of Clo-I coincide

with those of P-E, Clo-I must be the N-terminal

fragment. Results of CPase B digestion and hy-

drazinolysis of CPase B-treated P-E suggest that

Clo-‡W is the C-terminal peptide. The N-terminal

fragment H-1 gives overlap between Clo-I and

Vol. 91, No. 6, 1982


Fig. 4. Sequence study of P-E. Symbols used are as follows. •¨ Residue

identified as DNS-amino acid; ? residue which could not be identified as DNS-

amino acid; residue identified as PTH amino acid; ? residue which could

not be identified as PTH amino acid; ?? residue released by CPase B; ?? residue

released by CPase P; •` amino acid identified as a free amino acid when the residual

peptide after Edman degradation was analyzed on an amino acid analyzer. Arrows

above each residue indicate results obtained by Edman degradation or CPase diges

tions of intact P-E, while those under the residues represent results for the studies

with peptides derived.

Clo-‡U, since the total 9 mol of glutamic acid and

13 mol of glycine of H-1 were recovered in Clo-‡T

(Glu 6 mol, Gly 9 moll and Clo-‡U (Glu 3 mol,

Gly 4 moll as shown in Table ‡US. H-2 is the

peptide connecting three peptides as Clo-‡U-Clo-

‡V-Clo-‡W. This arrangement is consistent with

the observation for the amino acids released by

CPase P digestion of P-E.

Further confirmation of the C-terminal por

tion of the sequence -Lys-Ser-Arg-Ser-Pro-Arg of

P-E was obtained by the analysis of the peptides

released by trypsin from the CPase B-treated P-E.

Two peptides were detected on an amino acid

analyzer; one at the same position as that of Ser-

Pro derived from Clo-‡W by CPase B digestion,

and the other at the same position as that of Ser-

Arg (Clo-‡V).

These data suggest that the sequence of clos-

tripain peptides of P-E is Clo-‡T- Clo-‡U- Clo-‡V-

Clo-‡W. These sequence studies are summarized

in Fig. 4.

DISCUSSION

The supernatant fraction of parotid saliva treated with 80% methanol contained several basic pro-linerich peptides with very similar amino acid compositions.

The chromatographic pattern of parotto sauva

was different from that of whole saliva. In whole

saliva, P-B and P-C were the most abundant pro-

line-rich peptides (3) whereas in parotid saliva, the

contents of P-B and P-C were comparable with

those of other prolinerich peptides. One reason

for this difference is probably due to the contri

bution of submandibular and sublingual salivas to

whole saliva. Another reason may be the enzymic

modification of gland saliva components in the

oral cavity.

Levine and Keller (17) prepared basic proline

rich proteins from parotid saliva by ammonium

sulfate fractionation and chromatography on

DEAE-Sephadex A-25, Sephadex G-200, and SP-

Sephadex C-25. Although the preparative method

J. Biochem.

PAOLINE-RICH PEPTIDES OF HUMAN PAROTID SALIVA 2073

is different, it is probable that we are dealing with

the same peptides as theirs, since characteristics of

their proteins are very similar to ours.

Five peptides, P-B, P-C, P-D, P-E, and P-F,

were obtained in pure forms. Primary structures

of P-B and P-C were reported previously (3, 4).

The N-terminal portions of P-D, P-E, and P-F

show striking similarity, especially the complete

coincidence of the N-terminal 12 residues of P-E

and P-F. The amino acid composition and N-

terminal sequences suggest that P-C, P-D, P-E, and

P-F are very closely related peptides. The other

peptides, which were not well purified in this study, may also represent the same class of polypeptide

as P-C, P-D, P-E, and P-F, since their amino acid

compositions have common characteristics, such as

high content of glycine, proline, and glutamic acid,

and the lack of hydrophobic amino acid.

In the present work, P-E was further studied

for the determination of the complete amino acid

sequence. P-E was cleaved effectively by clostri

pain into four peptides. Elucidation of the struc

ture of the largest peptide, Clo-I, by subdigestion

with papain, an enzyme with broad specificity, was

unsuccessful, probably due to the presence of

repetitive structures in it. On the other hand,

manual Edman degradation of purified Clo-I pro

ceeded successfully when extraction of peptide was

prevented by removing trifluoroacetic acid prior to

the ethylene dichloride extraction. The reason for

the ambiguous result on the third residue of H-2

by the DNS-Edman method is not clear, but this

might be due to insufficient extraction of DNS-

arginine by ethyl acetate. Arginine was released

in a high yield from P-E by CPase B, in spite of

the presence of a penultimate proline. The high

release would be due to the relatively severe con

ditions used for the digestion (37•Ž, a ratio of

enzyme to substrate= 1 : 5 (mol/mol), 18 h). The

result of Edman degradation of H-1 indicates that

deamidation is incomplete in the conditions used

for mild acid hydrolysis.

The complete amino acid sequence revealed

that P-E was composed of 61 amino acids residues.

One of the remarkable structural features of this

peptide is the presence of the repetitive units.

When P-E is divided into the three domains ‡T

(1-21), ‡U (22-42), and ‡V (43-61) shown in Fig. 5,

each domain has homology of more than 50%

with the other domains. The homology between

domains ‡Tand‡U in fact reaches 90%. The longest

repeating unit is the sequence of 14 residues,

PPGKPQGPPPQGGN, which occurs twice in a

molecule. Other major repetitions are 3 occur

rences of the sequence PPGKPQGPPPQG, and 5

occurrences of PQGPP. P-E contains oligopro

line structures, -Pron-(n= 1-5) which were also

found in P-B and P-C (3, 4). It is also notable

that the C-terminal 7 residues are highly polar.

The close structural relationship between P-E

and P-C is further demonstrated by the fact that

they share the 16-residues sequence, PPPPPGKP-

QGPPPQGG, and the repetitive unit PQGPP. Since P-C is the C-terminal 44 residues of an acidic

prolinerich protein, Protein C (4, 18), P-E has a structural relationship with acidic prolinerich pro

teins.

Another basic prolinerich peptide, P-B, is

similar to P-E in being prolinerich, basic and

having oligoproline structures, but is different in

its relatively high content of hydrophobic amino

acids and the characteristics of the repetitive unit

(3).

Kauffman and Keller (19, 20) reported a se

quence study of the basic proline-rich protein IB-9. This protein is similar to P-E in its chromato

graphic behavior, amino acid composition, N- and C-termini, and partial amino acid sequences. How-

ever, they estimated its molecular weight as 9,000.

Therefore, whether P-E and IB-9 are identical still

Fig. 5. Repetitive sequence of P-E. Identical residues in domains are

enclosed in boxes.

Vol. 91, No. 6, 1982


remains to be clarified.

Genetic polymorphism of proline-rich pro

teins has been studied by Azen (21). A possible

reason for the occurrence of multiple forms of

proline-rich peptides might be genetic polymor

phism of precursor proline-rich proteins and their breakdown by enzymes. Because of the repeti

tiveness of precursor prolinerich proteins, their

enzyme-cleaved products would give complex chro

matographic spectra as observed in the present

study.Deamidation during preparation of peptides

cannot be excluded as a reason for polymorphism,

though there has been no example in the struc

tures so far determined.

REFERENCES

1. Ellison, S.A. (1978) in Saliva and Dental Caries

(Kleinberg, L, Ellison, S.A., & Mandel, I.D., eds.) pp. 13-30, Special Supplement to Microbiology

Abstracts, Information Retrieval, New York2. Keller, P., Levine, M., Sreebny, L.M., & Robino-

vitch, M. (1978) in Saliva and Dental Caries (Klein-berg, I., Ellison, S.A., & Mandel, I.D., eds.) pp.

547-555, Special Supplement to Microbiology Abstracts, Information Retrieval, New York

3. Isemura, S., Saitoh, E., & Sanada, K. (1979) J. Biochem. 86, 79-86

4. Isemura, S., Saitoh, E., & Sanada, K. (1980) J. Biochem. 87, 1071-1077

5. Sanada, K. & Isemura, S. (1980) Medicine and Biology (in Japanese) 101, 251-254

6. Spackman, D.H., Stein, W.H., & Moor.-, S. (1958)

Anal. Chem. 30, 1190-1206

7. Benson, J.V., Jr., Gordon, M.J., & Patterson, J.A.

(1967) Anal. Biochem. 18, 228-2408. Jackson, R.C. & Blobel, G. (1977) Proc. Natl.

Acad. Sci. U.S. 74, 5598-56029. Blakesley, R.W. & Boezi, J.A. (1977) Anal. Biochem.

82, 580-582

10. Muenzer, J., Bildstein, C., Gleason, M., & Carlson, D.M. (1979) J. Biol. Chem. 254, 5629-5634

11. Gray, W.R. (1967) in Methods in Enzymology

(Hirs, C.H.W., ed.) Vol. 11, pp. 139-151, Academic Press, New York

12. Iwanaga, S., Wallen, P., Groendahl, N.J., Henschen, A., & Blomback, B. (1969) Eur. J. Biochem. 8,

189-19913. Gray, W.R. (1967) in Methods in Enzymology (Hirs,

C.H.W., ed.) Vol. 11, pp. 469-475, Academic Press, New York

14. Woods, K.R. & Wang, K.T. (1967) Biochim. Bio

phys. Acta 133, 369-37015. Brenner, M., Niederwieser, A., & Pataki, G. (1969)

in Thin Layer Chromatography (Stahl, E., ed.,

Ashworth, M.R.F., translator) pp. 730-786, Springer-Verlag, Berlin, Heiderberg, New York

16. Hayashi, T. & Nagai, Y. (1980) J. Biochem. 87, 803-808

17. Levine, M. & Keller, P. (1977) Arch. Oral Biol. 22,

37-4118. Wong, R.S.C. & Bennick, A. (1980) J. Biol. Chem.

255, 5943-594819. Kauffman, D.L. & Keller, P.J. (1979) Arch. Oral

Biol. 24, 249-256

20. Keller, P.J., Kauffman, D.L., Bennick, A., & Wong, R.S.C. (1980) J. Dent. Res. 59, 541

21. Azen, E.A. (1978) Biochem. Genet. 16, 79-99

J. Biochem.


Supplemental Materials

TABLE IS Reaction conditions for sequence studies.

TABLE ‡US Amino acid compositions of P-E, clostripainpeptides

and mild acid hydrolysispeptides.

a H-1 represents a mixture of two peptides, one of which is lacking the C-terminal asoartic acid. See the text.

Fig. 1S Separation of clostripain digest of P-E. The

column of Bio-Gel P-4(1.3 •~ 160cm) was equilibrated and

eluted with 0.05M acetic acid. Peptides were detected by

following absorbance at 230 mm. Fractions of 2.4m1 were

collected. Clo-I was purified from the fractions 38-44

by chromatography with SP-Sephadex C-25 in the gradient

elution system of sodium acetate buffer containing NaCl.

Clo-‡U was purified from fractions 46-50 by the same

seethed.

Fig. 2S Separation of the mi1d acid hydrolysate Cf P-C.

The column of Bie-Gel P-6(1.7 •~ 130cm) was eguilrbratal

and elutedd with acetic acid. Peptides were detected

by following absorbance. at 230nm. Fraotions of 4.00m1

Vol. 91, No. 6, 1982

co11sctcd. rracGi ams 25-27 and G2-GS yielded fragments H-1

dar 11-2, respctireiy.

Documents

Fractionation and Characterization of Basic Proline-Rich