THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No . 27, Issue of September 25, PP. 15975-15981,1989 Printed in U. S. A.

Analysis of Imter-a-trypsin Inhibitor and a Novel Trypsin Inhibitor, Pre-a-trypsin Inhibitor, from Human Plasma POLYPEPTIDE CHAIN STOICHIOMETRY AND ASSEMBLY BY GLYCAN*

(Received for publication, April 19, 1989)

Jan J. Enghild, Ida B. Thegersen, Salvatore V. Pizzo, and Guy Salvesen$ From the Pathology Department, Duke University Medical Center, Durham, North Carolina 27710

The polypeptide chain composition of protein mate- rial referred to in the literature as “inter-a-trypsin inhibitor” was inves,tigated. The material was found to consist of distinct proteins of 125,000 and 225,000 Da, each of which contained more than one polypeptide chain. The links that assemble each protein were found to be stable to various strong denaturants, but suscep- tible to treatment with trifluoromethanesulfonic acid or hyaluronidase, indicating a glycan nature. The 225,000-Da protein. migrated with inter-a mobility on agarose gel electrophoresis and is designated inter-a- trypsin inhibitor, whereas the 125,000-Da protein mi- grated with pre-a mobility, and we designate it pre-a- trypsin inhibitor. Amalysis of the proteins, the sepa- rated chains, and proteolytic derivatives thereof re- vealed that each prlotein contained a single, identical, trypsin-inhibitory chain of 30,000 Da. Inter-a-trypsin inhibitor contains noninhibitory heavy chains of 65,000 and 70,000 Da, whereas pre-a-trypsin inhibi- tor contains a heavy chain of 90,000 Da. Our data allow identification of several recently reported cDNA clones and clarify tlhe confusion surrounding the com- position of plasma proteins referred to as inter-a-tryp- sin inhibitor.

The blood of mammals is a rich source of proteinase inhib- itors, accounting, in humans, for about 10% by weight of all blood proteins (Travis and Salvesen, 1983). These inhibitors control proteolysis that occurs during coagulation, comple- ment activation, fibrinolysis, and inflammation, but we are far from understanding the roles of several members of this proteinase inhibitor group. One of the most perplexing inhib- itors is inter-a-trypsin inhibitor (1aI)l (Heide et al., 1965), originally reported as protein H by Steinbuch and Loeb (1961). As indicated in a recent review by Gebhard and Hochstrasser (1986), little is known of the function of this inhibitor. Its primary structure has yet to be completely determined; in-

* This work was supported by National Institutes of Health Grant HL-24066 and American Cancer Society Grant IN-1588. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

P. 0. Box 3712, Duke University Medical Center, Durham, NC 27710. $ To whom correspondence should be addressed Pathology Dept.,

Tel.: 919-684-2412. The abbreviations used are: IaI, inter-a-trypsin inhibitor; PaI,

pre-a-trypsin inhibitor; TFMSA, trifluoromethanesulfonic acid; GAGS, glycosaminoglycans; TIC, trypsin inhibitor counterstain; PTH, phenylthiohydantoin; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; HC1, HC2, and HC3, heavy chains 1-3; HPLC, high pressure liquid chromatography.

deed, even its chain composition is unknown. Studies have revealed that the inhibitory activity of I d

resides in a fragment of about 30,000 Da (known as HI-30) that contains two domains, arranged in tandem, homologous to the pancreatic trypsin inhibitor (Kunitz) family of protein- ase inhibitors typified by aprotinin (for review, see Gebhard and Hochstrasser, 1986). The remainder of the molecule rang- ing from 150,000 to 220,000 Da is of unknown function. I d is thought to be composed of at least three chains that originate from distinct mRNA precursors (Kaumeyer et al., 1986; Schreitmiiller et al., 1987; Salier et al., 1987; Gebhard et al., 1988) and that are assembled by an unidentified mecha- nism. The links that stabilize the complex of the HI-30 and heavier noninhibitory chains resist dissociation in sosum dodecyl sulfate (SDS) in the presence of reagents that cleave disulfide bonds, leading earlier investigators to conclude that IaI is a single chain glycoprotein (Reisinger et al., 1985; Morii and Travis, 1985). Little is known of the nature of these links, although a brief report indicates that HI-30 is released from the parent molecule upon treatment with hyaluronidase or chondroitinase ABC (Jessen et al., 1988). As a prerequisite to investigating the function of I d , we have employed chemical and enzymatic methods to dissociate the molecule for analysis by inhibitory assays and protein sequencing, and we may now define the chain composition of this protein and others in human blood that contain HI-30.

EXPERIMENTAL PROCEDURES~

RESULTS AND DISCUSSION

Purification of SDS-stable Trypsin Inhibitors from Human Plasna-Plasma samples from blood drawn from human volunteers 15 min before SDS-PAGE contained SDS-stable trypsin inhibitors of 125,000 and 225,000 Da (Fig. 1). The trypsin inhibitor counterstain (TIC) assay was used to mon- itor fractions for SDS-stable trypsin-inhibitory activity dur- ing the purification of the human 225,000- and 125,000-Da proteins. The purification procedure was optimized to enable simultaneous recovery of both inhibitors from the same batch of plasma. We found that the 225,000-Da inhibitor bound to the blue agarose column under the conditions specified under “Experimental Procedures,” whereas the 125,000-Da inhibitor eluted during the initial column wash. The 225,000-Da protein eluted from the column in the 1 M NaCl wash. Although the 225,000-Da protein was very heterogeneous on ion-exchange chromatography, DEAE-Sephacel was found to provide a

Portions of this paper (including “Experimental Procedures” and Tables I-IX) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

15975

15976 Inter-a- and Pre-a-trypsin Inhibitors a b c d e f g h i

I -200

- 97 - 66

- 43 - 31

- 21 - 14

FIG. 1. Occurrence of SDS-stable trypsin inhibitors in hu- man plasma. Blood was collected from three volunteers from our laboratory. The cells were removed, and plasma, (2.5 pl (lanes b, d, and f ) and 5 pl (lanes c, e, and g)) was loaded on SDS-polyacrylamide gels within 15 min of collection. Lanes a and hare purified inhibitors, and lane i contains the molecular size standards (in kilodaltons). The 21,000-Da inhibitory band in lane i is soybean trypsin inhibitor.

a bed a b e d

190- 120- 97 - 66 - 43- 31-.

21 - - 14- 4

190- 120- 97- 66-

43 - 31 - 21 - 14-

FIG. 2. Dissociation of 225.000-Da inhibitor protein chains. SDS-PAGE is shown of standard proteins (lane a ) , purified inhibitor (lane b ) , inhibitor treated with 1 pg of hyaluronidase/25 pg of inhibitor for 3 h (lane c), and inhibitor treated with TFMSA (lane d) . Left, Coomassie Blue-stained gel; right, TIC-stained gel.

useful purification step. Most of the contaminating proteins eluted before the 225,000-Da inhibitor, which itself eluted rather late (0.4 M NaCl). Gel filtration on Sephacryl S-300 HR was employed as the last purification step, and the 225,000-Da protein was found to be homogeneous on SDS- PAGE with and without reduction, TIC gels (Fig. 2, lane b), and gel filtration on Superose 6.

As mentioned above, the fall-through from the equilibration of the blue agarose column contained the 125,000-Da trypsin inhibitor. The purification followed the same scheme as for the 225,000-Da protein. The 125,000-Da protein was not pure after gel filtration on Sephacryl S-300 HR, but separation on Mono Q resulted in pure material as judged by the criteria detailed above (Fig. 3, lune b) . 100 mg of pure 225,000-Da protein and 8 mg of pure 125,000-Da protein were obtained from 2 liters of plasma. Incorporation of the proteinase inhib- itors N-[N-(~-3-truns-carboxyloxiran-2-carbonyl)-~-leucyl]- 4-aminobutylguanidine, 3,4-dichloroisocoumarin, 1,lO-phen- anthroline, and EDTA during the purification did not increase the yield or stability of the proteins.

Identity of Inhibitors-Purified samples of each protein were electrophoresed in agarose gels as described under “Ex- perimental Procedures” (Fig. 4). Comparison with standard human plasma samples indicates that the 225,000-Da protein migrates between the a, and a2 zones and is therefore the previously characterized inter-a-trypsin inhibitor. The 125,000-Da protein migrates before the aI zone and may be identical to the prealbumin-like acid-stable trypsin inhibitor

a b c d

190- w 120- - - 97- bc 66- * 43- - 31- - 21- - 14-a

”

&

a b c d

190- 120- 97 - 66-

43 - 31 - 21 - 14-

FIG. 3. Dissociation of 125,000-Da inhibitor. SDS-PAGE is shown of standard proteins (lane a) , purified inhibitor (lane b ) , inhibitor after treatment with 1 pg of hyaluronidase/25 pg of inhibitor (lane c), and inhibitor treated with TFMSA (lane d) . Left, Coomassie Blue-stained gel; right, TIC-stained gel.

FIG. 4. Zonal agarose gel electrophoresis of purified pro- teins. Zonal electrophoresis was performed on samples of purified inhibitors. The gel was stained with Coomassie Blue and scanned at 600 nm. The broken line is the 225,000-Da protein, and the unbroken line is the 125,000-Da protein. The positions of the classic serum fractions are shown for comparison. Alb, albumin.

reported by 0dom and Ingwersen (1983) and the 125,000-Da IaI-like protein detected by Salier et al. (1980), although neither of these was characterized. In light of its migration in agarose gels and the relationship to I d , we name the protein pre-a-trypsin inhibitor (PaI).

Chain Composition-Plasma samples from blood drawn from human volunteers 15 min before SDS-PAGE revealed the presence of both inhibitors (Fig. l), indicating that Pa1 is not derived from IaI during plasma storage. Amino-terminal protein sequence analysis of the purified proteins revealed the presence of three chains composing IaI (Table I) and two chains composing Pa1 (Table 11). Sequence analysis of sam- ples analyzed by SDS-PAGE, after electroblotting on polyvi- nylidene difluoride membranes, did not show any change in the amino-terminal sequences, indicating that the multiple amino termini were not due to loose association of peptides or proteins. Treatment of the proteins with TFMSA as de- scribed under “Experimental Procedures,’’ employing a pro- tocol that is used to deglycosylate proteins, resulted in the production of 30,000- and 65,000-70,000-Da derivatives of IaI (Fig. 2, lane d ) and 30,000- and 90,000-Da derivatives of Pa1 (Fig. 3, lane d). Treatment of the proteins with hyaluronidase also resulted in the release of HI-30; yet under the conditions used for Fig. 2 (lane c), the 65,000-70,000-Da components of IaI remained associated on SDS-PAGE to give a derivative of 130,000 Da. The amino-terminal sequences of polypeptides released from IaI or Pa1 by TFMSA or hyaluronidase treat- ment were determined after analysis of the products by SDS- PAGE, as described under “Experimental Procedures”; and

Inter-a- and Pre-a-trypsin Inhibitors 15977

the results are presented in Tables 111-V. The 30,000-Da polypeptide from IaI was found to be identical to that of Pa1 and corresponds to the proteinase-inhibitory domain known as HI-30 (Gebhard and Hochstrasser, 1986). The 130,000-Da derivative of IaI produced by hyaluronidase treatment and the 65,000-70,000 derivatives produced by TFMSA treatment contained two amino-terminal sequences in approximately equal yields (Tables I11 and IV). One sequence was identical to residues Ser’-AspI4 of the IaI clone reported by Gebhard et al. (1988). The second sequence, inferred by subtraction from the first, does not correspond to sequences in release 19 of the National Biomedical Research Foundation Protein Identification Resource. The 90,000-Da derivative of Pa1 contained a single sequence (Table V) not found in this release of the National Biomedical Research Foundation resource. Therefore, the data presented in Figs. 2 and 3 and Tables III- VI1 enable us to conclude that IaI possesses two separate polypeptides designated heavy chains 1 and 2 (HC1 and HC2) in addition to HI-30, whereas Pa1 is comaosed of HI-30 and a 90,000-Da polypeptide designated heavy chain 3 (HC3).

The 65,000- and 70,000-Da derivatives of IaI were not well separated by SDS-PAGE, making it difficult to distinguish between them by sequence analysis following transfer to polyvinylidene difluoride membranes (see “Experimental Pro- cedures”). Nevertheless, we were able to designate the 65,000- Da derivative as heavy chain 1 and the 70,000-Da derivative as heavy chain 2.

Extended amino-terminal sequencing of heavy chains from I d and Pa1 was accomplished by treatment of samples with o-phthalaldehyde following the second Edman degradation cycle (see “Experimental Procedures”). This procedure pre- vents reaction of phenyl isothiocyanate with amino-terminal residues other than proline and allowed unambiguous identi- fication of extended sequences of the Pa1 heavy chain (Table VII) and the heavy chain of IaI that contains proline at position 3 (Table VI).

Sequence analysis of peptides derived from IaI (Table VIII) indicates that the heavy chains correspond to cDNA clones isolated by Schreitmuller et al. (1987), Salier et al. (1987), and Gebhard et al. (1989). Sequence analysis of peptides derived from proteolysis of Pa1 (Table IX) indicates that the heavy chain of this protein corresponds to the cDNA clone recently reported by Diarra-Mehrpour et al. (1989). The commercial anti-IaI antiserum used in our study does not recognize HC3 (Fig. 5), and we were not able to find any amino-terminal or internal protein sequence of HC3 associated with I d . There- fore, in contrast to the proposal of Diarra-Mehrpour et al. (1989), HC3 is not part of I d , but instead constitutes the heavy chain of PaI. It is therefore likely that the antiserum

TABLE X Amino terminal sequences of the components of IaI and Pal

Summary of results presented in the “Miniprint.” HI-30, also known as UTI (Gebhard and Hostrasser, 1986), contains trypsin- inhibitory activity. HC2 is identical to cDNA sequence reported by Gebhard et al. (1988). The amino-terminal sequence of HC1 has not been published; this chain corresponds to the partial cDNAs of Schreilmuller et al. (1987) and X Hu HITI-9 of Salier et al. (1987). The amino-terminal sequence of HC3 has not been published this chain corresponds to the partial cDNA X Hu HITI-13 of Diarra- Mehrpour et al. (1989).

Chain Residue number

1 5 10 15 20

HI-30 A V L P Q E E E G S G G G Q L V T E V T HC1 S K S S E K R Q A V D T A V D G T F I A HC2 S L P G E S E E M M E E V D Q V T L Y S HC3 S L P E G V A N G I E V Y S T K I N S K

a b c d e a bed e 7 ”_ - ’” 190- . .

190- - - c

r* 120- 120- - m

97- 66-

43- 31 - 21 - 14- -

97- - 66-

43- - 31- - L -

21 - .- 14- -

FIG. 5. Fai lure of commercial IaI antiserum to recognize Pa1 heavy chain (HC3). Samples of In1 or Pa1 (25 pg) were incubated with 1 pg of hyaluronidase for 180 min. Left, portions of 5 pg were analyzed by SDS-PAGE followed by staining with Coomassie blue; right, portions of 5 ng were analyzed by SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane for Western blotting with the commercial IaI antiserum as described under “Ex- perimental Procedures.” Samples were molecular size standards (lane a ) , untreated IaI (lane b), IaI treated with hyaluronidase (lane c), untreated Pa1 (lane d ) , and Pa1 treated with hyaluronidase (lane e ) . Note the reaction of the antiserum with all IaI chains, but the complete lack of reaction with Pa1 heavy chain.

190-

97 - 66 - 43 - 31 -

a b c d e f g h n h r

190- 120- 97 - 66-

43 - 21 - 31 - 14- 21 -

14 - /’

FIG. 6. Limited hyaluronidase treatment of I d . 25 pg of IaI was allowed to react with 1 pg of hyaluronidase for 5, 15, 30, 60, 120, and 180 min; and samples were analyzed by SDS-PAGE (lane c-h). Lane b is untreated inhibitor. Left, Coomassie Blue-stained gel; right, TIC-stained gel.

used by these authors to isolate the cDNA encoding HC3 was raised against a combination of IaI and Par. Table X sum- marizes the amino-terminal sequences of the chains compos- ing IaI and Pa1 and relates them to various published cDNA clones.

In addition to IaI and PaI, we purified a small amount (3 mg) of a 130,000-Da trypsin inhibitor that eluted slightly ahead of the IaI peak during blue agarose chromatography. Amino-terminal sequence analysis of this material revealed it to be composed of HI-30 plus HC2 (data not shown). We are not sure whether this material, with inter-a mobility, repre- sents a pre-existing HI-30 protein complex or is the result of dissociation of HC1 from IaI during plasma storage. Support- ing this latter possibility, we note that the HI-30/HC2 species accumulates upon storage of purified IaI.

Chain Stoichiometry-Since the relation between molecular size and migration of cross-linked polypeptides on SDS- PAGE is tenuous (Salvesen and Barrett, 1980), we were unable to infer the number of individual chains composing Pa1 a n d I d . However, limited hyaluronidase treatment of IaI resulted in a single molecular size shift to liberate HI-30 (Fig. 6); the remaining 130,000-Da derivative contained no inhibi- tory activity. More extensive digestion with hyaluronidase resulted in conversion of the 130,000-Da derivative into 65,000- and 70,000-Da derivatives, similar to TFMSA treat- ment, with no intermediates (Fig. 7). Extremely high hyalu- ronidase concentrations resulted in the appearance of addi- tional bands, most of which originate from the hyaluronidase preparation (Fig. 7, lane h). We conclude therefore that IaI

15978 Inter-a- and Pre-a-trypsin Inhibitors

190- 120- 97- 66-

43- 31-

21- I4-

O b c d e f a h a b c d e f a h

4

FIG. 7. Extended hya luronidase t rea tment of IaI. 5 wg of ItvI was incubated with 0, 15 ng, 30 ng, 90 ng, 0.2 pg, 1 f ig, or 5 pg of hyaluronidase for 180 min (lanes b-h); and portions were analyzed by SDS-PAGE. Lane b is untreated material. Left, Coomassie Blue- stained gel; right, TIC-stained gel.

a b c d e f q h a b c d e f q h

190-

97 - 66 - 43 - 31 - 21 - 14- I 14-

21 -

FIG. 8. Limited hyaluronidase t reatment of PaI. 25 pg of Pa1 was allowed t.o react with 1 pg of hyaluronidase for 5, 15, 30, 60, 120, and 180 min (lanes c-h); and samples were analyzed by SDS-PAGE. Lane b is untreated inhibitor. Left, Coomassie Blue-stained gel; right, TIC-stained gel.

contains single copies of two distinct 65,000-70,000-Da heavy chains (HC1 and HC2) and a single HI-30 chain. By similar criteria (Fig. 8), we conclude that Pa1 contains a single heavy chain (HC3) linked to a single HI-30 chain.

Despite earlier claims that IaI is a single chain protein (Reisinger et al., 1985; Morii and Travis, 1985) presumably due to the extraordinary stability of interchain cross-links, we present evidence here that IaI is composed of three chains and Pa1 of two. The first evidence for the composition of the cross-link was presented by Jessen et al. (1988), who showed that HI-30 could be separated from IaI by treatment of the protein with hyaluronidase and chondroitinase ABC, enzymes that share the ability to degrade chondroitin sulfate-like gly- cosaminoglycans (GAGS). However, Jessen et al. (1988) did not observe dissociation of the 130,000-Da derivative of I d , leading them to conclude that the molecule contains a single heavy chain. We show here that treatment of IaI and Pa1 with hyaluronidase or the deglycosylating agents TFMSA and trifluoroacetic acid leads to the liberation of all of the com- ponent chains of the proteins without detectable proteolysis, confirming the nonprotein nature of the cross-link and the chain stoichiometry shown in Fig. 9.

Location of Cross-link-Limited proteolysis of IaI using Staphylococcus aureus V8 proteinase results in cleavage of the Gl~’*-Val’~ peptide bond of HI-30 and the release of this chain from the molecule (Hochstrasser et al., 1981). In our hands, limited proteolysis of 101 with S. aureus V8 proteinase generated two fragments, stable to SDS-PAGE, with molec- ular sizes of 130,000 and 25,000 Da, respectively. Similarly, two fragments of 90,000 and 25,000 Da were generated from PaI. Amino-terminal sequence analysis revealed that both 25,000-Da derivatives correspond to the sequence from Val” of HI-30. Seauence analvsis of material followine: SDS-PAGE

HC I

HI-30 1 1 0 1

HC 2

HI-30 1 P O I

HC 3 ....

FIG. 9. Chain composition of IaI a n d PaI. This diagram shows the chain composition of IaI and PcrI. IaI is composed of two different heavy chains and one inhibitory light chain (HI-30), and the three subunits are probably held together with GAG (thin line). Pa1 is composed of one heavy chain, different from the two IcrI heavy chains, and one inhibitory light chain (HI-30), probably also linked together by GAG. The dots between the GAG chain and the protein chain represent the strong forces that assemble the complex.

Da derivative of Pa1 contain, in addition to their respective heavy chains, the first 18 residues of HI-30, all of which, except Ser’”, were positively identified. Since the amino- terminal 18-residue fragment of HI-30 remains attached to Ia I or Pa1 heavy chains in the presence of SDS, we conclude that the GAG-like cross-link originates from this region of HI-30, probably from Ser” since this residue was not detected unless the molecules were fully deglycosylated with TFMSA (Table IV). Furthermore, we note that the residues surround- ing Ser” conform to a partial consensus (DEXSG) for GAG addition to acceptor protein chains (Huber et al., 1988). Ser” of HI-30 has been reported to carry a long glycan chain by Hochstrasser et al. (1981), although these authors did not address the possibility of a GAG-like structure for the chain. Balduyck et al. (1986), on the other hand, identified a GAG- like glycan attached to human urinary trypsin inhibitor, a protein thought to be synonymous with HI-30 (Gebhard and Hochstrasser, 1986).

Preliminary data based on biosynthetic radiolabeling of Ia I in Hep G2 cells in the presence of [35S]sulfate indicate that HI-30, not HC1 or HC2, contains sulfated GAG (Swaim et al., 1988); and we reason that the GAG that assembles Ia I and, by inference, Pa1 originates from Ser” of HI-30. Since the IaI heavy chains are separated by extensive hyaluronidase treatment, we speculate that they are linked to each other by the GAG that originates from HI-30. Their greater resistance to hyaluronidase treatment suggests that the inter-heavy chain GAG is less available than the portion of the GAG chain that links HI-30 to the heavy chains. We know very little of the nature of this strong interaction between the HI- 30 GAG and the heavy chains, although Jessen et al. (1988) suggested that HI-30 is linked covalently to heavy chains of Ia I by chondroitin sulfate. However, in the absence of any direct evidence for a covalent cross-link, we feel that a strong, noncovalent association of HI-30 GAG with regions on HC1, HC2, and HC3 should also be considered. With respect to this last possibility, we note the report of Frenette et al. (1989) documenting a strong (stable to SDS-PAGE), but noncova- lent link between a heparin sulfate proteoglycan and laminin.

We believe that our observations clarify the confusion that has surrounded attempts to understand the structure of pro- tein material previously known as “inter-a-trypsin inhibitor.” We have shown that the material consists of two distinct complexes, each containing a single trypsin-inhibitory chain that assembles, via a GAG-like glycan, with either HC3 to give Pa1 or with HC1 and HC2 to give IaI. An understanding of the structure of the inhibitors should enable the design of experiments to examine the function of the inhibitors and the reasons for their unusual glycan-mediated assembly.

revealed that the 130,OOb-Da derivative of Ia I a id the 90,000- Acknowledgments-We thank Jan Potempa, James Travis, Wil-

Inter-a- and Pre-a-trypsin Inhibitors 15979

liam Wagner, and John Mort for helpful discussions, Wolfgang Geb- hard for communicating results before publication, and Pat Burks for typing of this manuscript.

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SUPPLEMENTAL MATERIAL TO

ANALYSIS OF INTFR-0-TRYPSIN INHIBITOR XND A NOVEL INHIBITOR PRE-o-TRYPSIN INHIBITOR. FROM HUMAN PLASMA

BY

Jan J. Enghlld. Ida 8. Thggerren. Salvatore V. Plezo and Gu) Salvesen

EXPERIMENTAL PROCEDURES

TABLE I:

Cycle NO.

I8 E L F 19 V Y I 20 T S A

95. 235. 133 96. 148. 99 . , 22 ~~ ..

Inter-a- and Pre-a-trypsin Inhibitors TABLE 11: AMINO-TERMINAL SEQUENCE OF Psi " ~~ "_ "_

C7Ek PTH NO.

Net

(P mall Yield

". . "~~ "" " . ~~~ ~

1 ,\ s 162. .- 2 V L 3 L P

146. 205 ... 111

TABLE VI? A M I N O ~ T E ~ M I N A ~ SEQUENCE OF 1-1

TREATED WiTH OPA IH CYCLE 3

~

Cycle PTH NO. Yield

(P moll

Net

_ _ _ ~ ." .__ . "~ . ..

91. 76 -.* 5 1

3 b 77 19 ... 12

78. - - 37

17, 12 -, 37

31, ~~

--, 23

21, - 28. 11 ", 11 8. 1

18, -- ", 6

AS Y L K i4Z1. 561. ,329

2108, --* P R O O G 107

I 1 3 i 8 9 10 I1 12 13 24 I S 16 17 I 8 19 20

5 6 7 B 9 IO

C S E 233 E U 150

E 230 n

C'E

G Y C Y

Q S I, T V K

E N T i

T K v s

, I 12 13 14 15 16 I t 18 I 9 20

61 193

TABLE 111: AMINO-TERMINAL SEQUENCE OF HYALURONlDhSE-TREATED

1111 DERIVATIVES

.. ~ ~ " ~ . . . ~- ~~

130.00 D. Doriue.tive 30,000 Da Derivative .. ~~~~ ~- . . ~ ~

Cycle PTH Net

Yield PTB Net

Yield NO. (P moll (P mol)

TABLE Wr: AMINO-TERMINAL SEQUENCE OF Pal TREATED

WIT11 OPA IN CYCLE 2

. . . -~ ~ ~ ""

Cycle PTH NO.

Net

(P moll Yield

A S V I.

." - ~~ . 1198. --a 951, 830

P 505 E 215 c 392 V 653

a Y I, P Q E e E

:,b G

102 117

102 131 20 13

2 3 1 5 6 7 8 9 IO I1 12 I 3 I 4 I S

R 9 i o 11

163

98 G

13 14 15 16 11 I 6 19 20

G Q 95 L 1 1 Y 98

16 K 11 I 18 N 19 S

I44 183 93

99

"

T E 56 V 68 T

TABLE nil. PEPTIC PEPTIDES DERIVED PROM 1ai TABLE Iv: AMINO-TERMINAL SEQUENCES OF TPMM-TREATED 101 DERIVATIVES

Peptide i Peptide 2 Peptide 3 Pepme 4 Peptide 5

Cycle PTH Net PTH Net PTIK Net PTH Net PTH Net NO. Yield Yield Yield

(PrnOlI tpmoll Yield

lpmoll (pmol) Yield Ipmoll

L T --b L I 17 I 29 I 180 F 438

Cycle PTH Net NO.

(P mol) Yield

275 I i Y P D T F B G H

T K c Q V \ D h K 8 Y 1 E

S "/a

P 46 S

I06 U 91 T

Y U G K

V 11 F P A P D N L D P I

516

216 ID6 11

0

i s 1 2 3

\ i' I,

239 1 7 2

107 255 310 240 5 9

32 55

Q E E E G s G G G Q i, V

4 L R

256 182

3 7 32 25

35 31 IS

82 53 55

69 50 119

5fie 274

11 2

is 14

Y 1 4 s so

46 34

42

13 154 100 201

34 11 E

K 15 I 135 P 26 K 9 16 Q 104 V 34 I 2 4 17 P 96 I 19 Q I 1

128 132 135

18 s -- s - - 19 G 94 K 5 S ..

P 13

20 G ~~ G 18 G -- 1 7 i 6 19

T p. v

14 38

2 1 22 23

9 7 Q -- K 3

TABl.8 Vr AMINO-TERMINAL SBQUENCE DP ItYALURONIDASE-TREATED

Pal D ~ R I V A T I V ~ S

-~ . . . ..

90,000 Da Del i~St ivC 30,000 Ds Derivative . . . . . . . . ~. .

Cycle PTH Net PTH NO. Yield Yield

tp meit tp moll

Net

~ ~ ." . . ."

Inter-cu- and Pre-a-trypsin Inhibitors

x ~~ I 133

P r i' c V T I > K S 9

i L K Y Y a N A I K C R Y

i' Y N I. c F

20

c l a s -- s - - n 3

Y 5 1: 6

15981