Hydrolysis of peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease

.kRCHIVES

OF

Hydrolysis

RIOCHEMISTRY

.\ND

of Peptide

BIOPHYSICS

Bonds

Endothia DON C. WILLIAMS, Department of Biology, and Department

JOHN

149,

52-61 (1972)

of the Oxidized parasitica

It. WHITARER2

B-chain

of Insulin

Protease’ AXI)

PAMELA

V. CALDWELL

Western Washington ,State College, Bellingham, Washington of Food Science and Technology, University of California, Davis, California 95616

Received

September

9, 1971; accepted

by

November

98225,

26, 1971

The relat,ivc rates of hydrolysis of the peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease have been determined at pH 3.60 and at a suhof 1.95 X 10m6Y. strate concentration of 1.07 X 1OW M and an enzyme concentration The Pheza-Phere bond is hydrolyzed at a maximum rate followed by hydrolysis of the Tyr10-Leu17 and Gina-Hiss bonds. The Leull-VaIla and Asna-GlnA bonds are hydrolyzed at slower rates. The Leull-Vallz bond appears to be considerably more resistant t,o hydrolysis in the peptide 5-16 than in the inlact oxidized B-chain. The Leulj-Tyrls bond is very slowly hydrolyzed in peptide 5-16 but there is no evidence for hydrolysis of this bond in the int,act oxidized B-chain. Phexj is slowly hydrolyzed from the peptide 25-30 and the bond involving Gly~Gluz~ is slowly hydrolyzed in peptide 12-24 and/or peptide 17-24. Thus the enzyme has specificity for the hydrophobic regions of the oxidized B-chain. The specificity of Endolhiu parasitica protease on the oxidized B-chain of insulin is similar to that, of pepsin in that bonds involving Gln-His5 , are hydrolyzed Leull-Vallz , Leulj-Tyrls , Tyrlc-Lelllr . Phesr-Phess and Phetj-Tyrzs by both enzymes. Endothia parasitica protease hydrolyzes bonds involving Asnt-Glnd and Gly,,-Gluzl , not attacked by pepsin, but fails to hydrolyze bonds involving Pheland Gly23-Phet4 , hydrolyzed by pepsin. Thus, Endothia Valz , Gluls-Alald , Alala-Leulj parasitica protease is more specific in its action on the oxidized B-chain of insulin than 1s pepsin.

A protcolytic enzymcb produced in large quantities by Endothia parasitica has been purified and crystallized (1, 2) and a number of its chemical, physical and enzymatic propcrtics studied (l-5). Thr enzyme rapidly clots milk at’ pH 5-G and hydrolyzes cascin and hemoglobin at maximum rates at’ pH 2-2.5. It is not inhibited by sulfhydryl rc:Lgents, metal chelating reagents or by tosyl-L-phcnylalaninc chloromethyl k&one (TPCK) or tosyl-I,-lgsine chloromct’hyl ketone (TLCB, 1). The enzyme appears to b(4ong to the group of acid proteases. No work has been published on t’hc substrate specificity of this enzyme although it is 1 This work was supported in part by the National 1nstitut)es of Health (5 ROl i\M13165). 2 To whom reprint requests should be direct,ed at) the University of California.

Cog,yrigllt

@ 1972 by .\citdcrnic

Fress,

Inr.

known to cause clxtcnsivc fragmentation of the oxidized B-chain of insulin (3). The oxidized R-chain of insulin is a convcnicnt substrate for determination of spccificity of protcolytic enzymes since its amino acid scc~uc~m: is known (6). The oxidized Hchain has bwn used for studying the spcG ficity of a number of protcolytic enzyme including trypsin (6), chymotrypsin (6)) subtilisin (7), clastase (8, 9), papain (lo), chymopapain (10, ll), papaya peptidase (lo), rennin (12)) pepsin (6), pepsin C (13), Mucor miehei prot’easc (14)) streptococcal protease (15), plasmin (l(i), thermolysin (17) and Bacillus subtilis ncwtral protcaso (IS). In the majority of casts for substrate specificity determinations, the oxidized R-chain was incub&d with cxnzymr: until csscntially compl(>to hydrolysis of a11 watlily suscopt~iblo

E. PAZIASZTICA

PROTEASE:

BCTION

bonds w-as obtained. One cxccption is t!he elt>gant work with t’rypsin where the rates of hydrolysis of pcpt’ide bonds involving the arginyl and lysyl residues of the oxidized Bchain were determined (19). This invest.igation was greatly facilitated since only two bonds arc hydrolyzed by trypsin. It, is well-kno\vn that the suscept~iblc bonds of protein substrates and synthetic substrates arc hJ;drolyzed at different’ rates. At pH S, trypsm hydrolyzes the Arg22-G1y2s bond of the oxidized B-chain of insulin 25 times faster than the Lyszs-Alaao bond (19). This difference becomes more pronounced at higher pH values. Chymot,rgpsin has maximum specificity toward bonds involving t,yrosine, phwlylalaninc and tryptophan but will also slowly hydrolyze bonds involving ot(hcr amino acid residues. For example, li,,t values for hydrolysis of N-acetyl dcrivativcs of L-tyrosine ethyl ester, L-phcnylalanine c%hyl (~stcr, L-tryptophan c%hyl ester, Lvalinr methyl wtcr and glycine methyl ester are: 193 (20), 173 (al), 50.6 (20), 0.15 (22) and 0.00s WC-~ (22), rcspcctivcly. l’rptide :rnalysw following various degrees of hydrolysis of the oxidized B-chain of insulin pclrmits a drtcrmination of the relative rates of hydrolysis of the ptptidc bonds of that substrate. Other advantages of doing timcscquc~m studies include unequivocal dctcrmination of whether E’ndolhia parasitica prokasc is an (Indo- or cxopeptidasc, the effect of nonadjacent amino acid residues on rate, and evaluation of possible transpeptidat’ion reactions. These advantages are offart by the large amount of work involwd in separating and determining the idcntit’y of pcptidw formed at various time intervals. Tht, following report conwrns a determinat,ion of the peptidc bonds of the oxidized B-chain of insulin hydrolyzrd by E&~thia parasitica protease as a function of timcl and extent’ of hydrolysis. EXPEI‘LIMENTAI, Materials.

Endolhia

P11OCEI~IJI:.E parasitica

protease

was

purified by the procedure of H,zgemeyercf al. (1). Insulin (lot 100-1600, activity 24 IU per mg), DEAE-Sephadex ,450.120 (lot 48B-0550-1) and 2,4,&trinitrobenzene sulfonic acid were obtained from Sigma Chemical Co. Dow-ex 5OW-X4 (analytical grade AG50-X4. 100-200 mesh, lot 405&41

ON OXII~IZEI)

B-CHAT?i

OF INSULIN

53

H-29(i) was from Rio-Itad 1,aboratories. Ninhydrin and hydrindantin were from Nutritional Biochemical Corp. Pyridine was analytical reagent material from RIallinckrodt All other compounds were reagent grade. ,Velhods. Oxidized B-chain of ins~clin. Insulin was osidized by the procedure of Craig et al. (23). In a typical experiment, 0.200 g of insulin was dissolved in 10 ml 98$;, formic acid and cooled to 0” in an ice bath. Eight,eenmilliliters of formic acid and 2 ml of 30:; IILL were combined and after standing for 2 hr the solution was added to the insulin solution. After a 2.5.hr reaction, 400 ml of water was added and the solution was lyophilized to give 0.192 g oxidized A and B chains. The oxidized A and B chains were separated by the method of Bang-Jensen et al. (12) using a 3.0 X 12.5 cm column of l)EAE-Sephadex A-50-120 and pyridine acetate buffers. The cluant from the column was monitored by a ninhydrin method (24). Fractions containing the individual peptide chains were pooled and lyophilized. Enzyma(ic ky&olysis. Oxidized B-chain of insulin (0.1800 g) was dissolved in 2.25 ml of 50y0 formic acid and mixed wit,h -45.0 ml of 0.55 M (with respect to a&ate) pyridine acetate buffer, pII 4.80. The pII was 3.GO. It was necessary to use the above procedure for dissolving the substrate because of its slow solution in pyridine acetate buffer at pH 3.6. After equilibration at 30.0”, 3.75 ml of the reaction mixture was removed into 0.67 N HCl. To the retnaining solutiott 1.0 ml of Endolhia parasitica protease (3.2 mg/ml) was added. The reaction contained 1.07 X 10el M oxidized B-chain and 1.95 X 10-G M enzyme (molecular weight of enzyme = 37,500; 500-fold ratio of substrate to enzyme). At intervals 3.87ml aliquots of the reaction were removed into 0.07 ml li K HCl and Iyophilized. At the same intervals, duplicate O.O(i-ml aliquots were removed into 1.0 ml 2’,, NaHCOa , pH S.5 for deierminat,ion of increase in amino groups by the trittitrobenzene sulfonic acid method (25). The conc.entration of amino groups were calculated using a molar extinction coefficient of 1.3 X 10” M-I cm-l (‘15). At the end of 2-2 hr of incubation a new aliquot of enzyme was added to the reaction and I he incrlbat ion continued for another 2-l hr. Separalion and idenlijkalion of peptides. Lyophilized digests from the enzymat,ic hydrolysis of the oxidized B-chain were dissolved in 0.5 ml of 0.2 M pyridine acetate buffer, pH 2.3 and placed on top of a 0.9 X GO cm column of I)owex 50-X4 equilibrated with 0.2 M pyridine acetate buffer, pH 3.1 (10). Before use, the Dowes 50-X4 was purified as described by Moore and co-workers (2G, 27). .4fter use, the column was regenerated

34

WILLIAMS,

WHITAKER,

with 200 ml of 0.2 M NaOH followed by 600 ml of 0.2 M pyridine acet,ate buffer, pH 3.1. For elution of the peptides, the column was washed with 0.2 51 pyridine acetate buffer (135 ml) followed by a linear buffer gradient increasing to 2.0 SI pyridine acetate buffer, pH 5.0 (500 ml). The column was then washed with 100 ml 2 i~fpyridine acetate, pH 6.6. No peptides were eluted in this latter buffer. The flow rat,e was kept constant at 25 ml/hr by use of a Milton Roy Minipump. Three-millilit,er fractions were collected and analyzed for peptides by the ninhydrin method (24). Fract.ions containing a peak of ninhydrin-positive material were combined, lyophilized and hydrolyzed in 6 N HCl for 70 hr at 110” (28). Amino acid analyses n-ere performed on a Technicon Auto-Analyzer. RESULTS

AND CALDWELL TABLE

1

HYDROLYSIS OF OXIDIZED B-CH.\IN UE‘ INSULIN EXDOTIIIA PARASITICA PROTLISV Hydrolysis time

Bonds Ilydrolyzed per mole substrate (avg)

Bonds l~ydrolised per mole Yxbet rate (avg)

~...__~_~ I mm)

IWLitL)

-0.25 1.0 2.0 5.0 10.0 20.0

lTgdrolysis time

UY

0.28 0.76 0.88 1.34 1.70 1.85

60.0 120 240 1440 2880b

2.30 2.57 2.96 4.24 4.84

u Increase in amino groups determined by the trinitrobenzene sulfonie acid method as described in text. The reaction contained 1.07 X lo-” 11 oxidized B-chain and 1.95 X 1OP Y enzyme at pH 3.60 in 0.51 M pyridine acetate buffer containing 2.37(, formate. Incubation was at 30.0”. * New aliquot of enzyme identical t.o original was added at 1440 min and inmhntion contiluned to 2880 min.

The rate: of hydrolysis of thr oxidized Bchain of insulin by Endothia parasitica protease under the condit8ions used as dttermined by t,he trinitrobenzenc sulfonie acid reagent is shown in Table I. Experimental conditions were cl~osen on t,hc basis of prior experiments so that t’he rat’<: of hydrolysis would be slow enough to permit samples to formed. The combining rat,ios of tZhclamino be t&en near t,hr* beginning of hydrolysis acids obtained from each sample analyzed and yet fast enough to eswnt’ially complete arc shown in Table II and an identificat,ion the reaction lvithin 48 hr. As shown, hydrolbased on the known sequence of the oxidized ysis was quite rapid war t,hr beginning with R-chain of insulin (Fig. 3) is giwn. an average bond splitting of 0.2s approxiBefore t,rratment with cnzym(‘, the oximately 0.25 min aftw beginning the rcaetion. dized B-chain gave a single pwk on Dowex ;$t, the end of 1 min 0.76 bonds \wrcb hydro50-X4 (Fig. 1). The bascllinci increase due to lyzed. This valw approximately doubled ninhydrin reactive material following fracaftrr 5 min and again after about 120 min. tion no. 115 is t,ypical of this elution schrmc. At the end of 24 hr (1440 min) an average of As shown in Table II, the d(+crmint~d amino 4.24 bonds were hydrolyz~~d. Addition of a acid composition of this pc:ak agreed ~11 new aliquot of enzyme at 24 hr followId by n-it,h the know-n amino acid composition of an addit,ional 24-hr incubation pclriod in- the>oxidized R-chain of insulin. Following waction of tlw oxidizcxd B-chain creased the bonds hydrolyzed by 0.60 to a final vnluc of 4.84. with B. pa.rasitica. protease for approximately Separation of the pcptides formed after 0.2,5 min thrw was an av(wg(’ bond splitting various intc>rvals of incubat,ion is shown in of 0.2s (Table I) and tlw formation of small Figs. 1 and 2. The fractions indicated by the amounts of six peptidcs. Tllcl Phepl-Plw~~, bars at the bottom of each diagram w-cre TyrlG-LeulT and Leull-Vnlls bonds wcrc split pooled, lyophilizcd and hydrolyzed with 0 N in the proportions of approsimat~c~ly 1, 1 and O.S, rc~spcct~iwly, bawd on t ltc artw under HCl for amino acid analyses. Th(x fractions t81ictcurves in P’ig. 1. (Tlw :uws arc 0nlJ combined were chosen so as to lsrgr~ly climroughly proport’ional to conwntrution bcinatn cont~amination by pcptidw in adjnccnt cause of large variation in color yield among peaks. This proccdurc facilitated idcntification of the: major pcptidcl pwsent in a peak peptidcs.) Peal; no. 11 n‘as :i mixtuw of pepbut it, prwented a quantitative nnulysis of tides l-11, 17-30 and 23-30. Tlw expwtcd cnch pcptidr present and may baw pre- peptidc l-16 (from hydrolysis of TyrlB-L(wly bond) was not found. According to tlw chrowntc>d dctrct>ion of some minor pcptidcs

0.8

r

q

before

hydrolysis

t 0.6

t

0.4

/ $

0.2

2 0

E ::

0.25

0.8

minutes

2 0.6

1 0.4

0.2

I 180

0

FRACTION

NO.

60

120

180

(3.0ml)

FIG. 1. Peptides produced from the limit,ed action (O-5 min) of E’ndolhia ~~~usilicu protease on the oxidized B-chain of insulin. Reaction conditions were: 1.07 X 10-S M substrstc and 1.9 x 10-C M enzyme at pH 3.60 in pyridine acetate-format,e bluffer. Peptides separated on a 0.9 X 60 am colmnn of Dowex 50-X4 and the eluant morli(ored by a ninhydrin method. F’eptides identified from amino arid composition (see Table II).

r

1440

minutes

2880

minutes

0.9

0.6

:i

120

minutes

1

0.9

0.6

0

0

60

120

180

FRACTION

FIG. 2. Peptidcs B-chain of insulin.

0

NO.

produced from t’he action of Endofhia Conditions BR in Fig. 1. 55

60

120

180

(3.0ml)

parasitica

protexse

on the oxidized

TABLE PEPTIDES

PRODUCED - -

..--

II

BY ENDOTHIAPARASITICA PHOTEASE OF INSULIN _____--

ACTION

OK OXIDIZED

B-CHAIN --__--_

Time

Peak

(min)

Eo.~

Cysteic

Asp

Thr

Ser

Glu

Pro

Cly

Ala

Val

Leu

Tyr

Phe

Lys

His

Arg

0

7

2.0 (2jC

1.0 (1)

0.8qb (1)

0.8qb 2.7 (1) (3)

1.0 (1)

3.2 (3)

1.9 (2)

2.9 (3)

4.1 (4)

l.ab (2)

3.0 (3)

1.0 (1)

1.9 (2)

1.0 (1)

original substrate

0.12

+.$

+$

0

2.5 (3)

1.3 (1)

3.2 (3)

4.4 (4)

0.59 (1)

2.1 (2)

0.16 2.7

0.47

81-24

2.7 (3)

1.2 (1)

2.2 (2)

1.1 (1)

original substrate

TLO.25

2.0

5.0

Combining

4

c 1.6 (1)

1.2 0.90 0.83 -____-__--------(1) (1) (1)

2.7 (3)

0.83 3.5 (1) (3)

2.2 (2)

3.3 (3)

4.6 (4)

2.2 (2)

g

0.42 -(1)

0.23 0.64 m-

0.24

0.76 (1)

0.92 (1)

1.2 (2)

0.98 1.1 (1) (1)

-1.2 (1)

0.87 1.1 __(1) (2)

0.76 (1)

0.32 0.45 (1)

1117-30

14

0.96 (1)

0.16 0.72 (1)

0.20

1.1

1.3 (1)

2.1 (2)

--1.2 (2)

1.8 (2)

1.8 (2)

--1.4 (2)

1.0 (1)

0.48 0.84 (1)

1112-30

4

--2.2

1.0 (1)

-1.1 (1)

3.00 (3)

3.0 1.1 _---__(3) (1)

3.3 (3)

4.4 (4)

0.89 2.1 (1) (2)

0.16 -~ 2.3 (2)

0.94 (1)

111-24

7

2.6

0 85 0 65 LL (1) (1)

0.95 (1)

3.2 (3)

1.0 (1)

5.2 (3)

--1.7 (2)

3.3 (3)

4.8 (4)

2.2 (2)

4.0 (3)

0.65 1.4 __(1) (2)

1.8 (1)

original substrate

9

0.72 (1)

0.21 fg

0.20

0.82 (1)

1.5 (1)

1.3 (2)

1.6 (1)

0.89 1.2 (1) (1)

__0.52 (1)

1.2 (2)

-1.4 (1)

0.43 0.41 (1)

117-30 (some contamination by BZS-30)

12

0.28

0.09 0.86 (1)

0.09

0.38 1.1 (1)

0.60 1.1 (1)

--1.2 (1)

1.0 (1)

0.14 0.24

1125-30 (some contamination by 1117-30)

4

2.7

1.2

0.87

0.85 3.1 (1) (3)

3.7 (4)

1.0 (1)

2.1 (2)

2.1

11.

81-24

(1)

G (3)

0

(1)

2.8 (3)

0

(2) 0.65

0.41 0.18

0.64

1.1 (1)

0.24

1.3

1.3

1.2

2.5

0.99

0.86

0.24

1.0

0.32

15-16

(1)

(1)

(1)

(3)

(1)

12

(2) (2)

0.05

0

(1)

(2)

0.36 0.44 1.1 (1)

1.8 (2)

(2) (1) (2)

0

0.03 0.89 (1)

0.04

0.10 1.1 (1)

0.18 1.0 (1)

4

2.1 (2)

0.970 (1)

0.79 (1)

2.9 (3)

4.4 (3)

8

0.70 (1)

0.38 0.06

0.65 (1)

0.64 0 (1)

0

0.02 0.9~ (1)

0.05

0.07 0.98 (1)

0.10 1.0 (1)

4

0.92 (1)

0.43 0.08

0.12

1.7 (1)

Q (2)

8

0.71 (1)

0.23 0.06

0.50 (1)

0.89 0 (1)

0.92 0.94 0.91 (1) (1) (1)

12

0.04

0.04 0.84 (1)

0.07

0.42 j& (1)

0.210.92 (1)

0.07 0.19 0.58 1.1 (1) (1)

0.80 (1)

0.15 0.02

0.05

1.0 (1)

1.5 0

0.12 --0.94 1.0 (1)

(1)

1.2 (1)

0.04 0.44

_2.0

1.0

2.5

1.2

0.58

0.96 0.31 0.34 0.82

(2)

(1)

(2)

(2)

(1)

(1)

0.28

0.64 (1)

12

1440

(2) (1)

f$

(1)

120

c"nclIIsiolls

7

8

20.0

ratios

2 3 5

0.03

0 0.08

0

0

0

2.30

(2)

0.42 0

0.04 0.13 0.60 1.0 (1) (1)

0 . 74 3 . 3 (1) (3)

1.0 (1)

0.05 0.12

4.0 (4)

1.3 (1)

2.3 0

0.97 1.4

0.87 3.4

1.4

0.41 0.24 1.3

(1)

(1)

(1)

(1)

0.05 0.10 1.0 (1)

0.65 2.0 (1)

0.46 0.45 1.1 (1)

56

(3)

0.06 --1.6

(2)

(125-30

1.5 (1)

11-24

0

115-16

(2)

1.0 (1)

0.98 0.06 0.02 (1)

1125-30

1.1 (1)

0.26 1.2 (1)

0.12 0.26 0.82 (1)

f/17-24

3.2 (3)

1.1 (1)

0

0.35 0.06 -1.6

(2)

1.6 (1)

0.83 O.lll.0

1.1 (1)

0.05

0.19 0.11

1125-30

0.04 0.29 0.76

1117-24

(1)

112-24

(1)

0.09 0.25 0.15 (1)

115-16

11-3

E. PAR~lSII’ICA

PBOTI~:ASE:

ACTION TABLE

..

~. _..~

..

Tilnr

Peak

(mid

NO."

Cysteic

8

&g

ON OSII~IZED

R-CHAIN

II (Continued)

~.--~------_.---___-__ Combining Thr

Ser

Gl"

Ala

0.20 0.08

0.78 ____ (1)

0.78 0 (1)

--1.1 (1)

0.90 0.92 4.1 --__(1) (1) (3)

0

0.01&O. (1)

0.07

0.06 1.1 (1)

0.13 Q (1)

1

1.3 (1)

0.40 0.07

0.79 __(1)

1.0 (1)

0

G (1)

0.81 0.82 4.3 I_-~(1) (1) (3)

0.05 0.40 0.09

2

0.81 T

0.18 0.02

0.07

&J (1)

0

G

0.12

1.1

1.1

0

2.1

3

0.94 (1)

0.92 0.03

0.08

?_li

0

--1.9

1.2

3.9

1.6

5

0

!I.80 (1)

0.02

0.04

0.37 0

0.28 0.39

8

7-F 0.84

0.23 0.10

0.72 (1)

0.70 0 (1)

--1.0 (1)

1.4 (1)

10

&-8I (1)

0.06 0.05

$.$

0.11 0

G (1)

0.15 0.12 z.0

12

0

0.03 o.ss (1)

0.38

0.28

0.34

12 (1)

13

0

0.01 -1.0 (1)

0.14

0.07 G (1)

15

0

0.01 0.17

0.05

0.82 (1)

Asp

(1)

2880

(2)

Pro

Conclusions

ratios

Glv

13

57

OF INSULIX

(2) (2)

LO (1)

(1)

Val

I.eu

0.17 2 (1)

Phe

Lys

His

-1.5 (1)

0.78 0.08 &j

Airg

(2)

15-16 #26-JO

1.2 m

0.03

"5-15

0.08 0.37 0.79

__ (1)

l/17-24

0.87 1.0

0.05 0.06 0.94

1112-24 (some ill-3)

1.2 (1)

0.99 0.10 G (1)

0.04 0.26 0.10

>/l-3

0.76 __(1)

4.7 (3)

0.25 -1.5

0.07

ii5-16

0

115-11

0.19 Q (1)

(1) (1)

1.1 m

0.03

0.15 0.05

0.04

0.31

(1)

(1) (1)

(2) (2)

2-1 (1)

1.4

m-

(2)

0.86 0.83 0.12 G

(2)

(2)

0.14 0.82 (1)

Tyr

0.12 0.88 l.D (1)

1.0 (1)

0.93 __(1)

0.81 0

0.04 0.23 _0.92 0.15 _0.91 0.19 0.10 (1) (1)

0.18 0.08 0.10 0.32

0.19 0.09

1.0 (1)

1.1 (1)

1125-30 1126-30 !121-24 ---

a b

Numbers are those The values

for

used in Figures

serine,

tbreonine

l-2

and are assigned

and tyrosine

on basis

of order

are not corrected

for

eluted

front column.

degradation

during

the 70 hr.

hydrolysis

procedure. c d

Values

in parenthesis

Underlined

anxino acids

indicate considered

theoretical

composition

to be derived

of peptide.

from the major

peptide

S03H 5 10 I Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu~Tyr-Leu-Val-Cys-GlyT ? 7 25 -Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala r FIG. 3. Amino acid sequence of the oxidized by Endothia parasitica protease. The thicker hydrolysis.

matographic data of Rich& (14) peptide l-16 would be eluted at the same position as the oxidized B-chain. Wherever identical peptides were formed in this work and that

present.

S03H 15

I :

T

20 At I

30

? R-chain of insulin (6) and points of hydrolysis the base of the arrow, the faster the rate of

of Rich& on Mucor miehei proteasc identical chromatographic behavior was observed except for peptides 25-30 and 26-30. In our work peptidc 25-30 is clearly eluted befort>

5s

WILLIAMS,

WHI’I’AKI~:R,

p(ytid(a 2-30 (Fig. 2, 1440 and 28X0 mitt) \vltil(~ IZiclwt reporkd tlw oppositcl b+ Itavior. Aftw I mitt reaction time with an average bond splitting of 0.76 tltcrc> was a furthw incwaw in thca amounts of peptides l-24 and 17-30 (data not, sho\vn). l’rptidc 12-30 was not) id(wtificd althouglt it appcwcd t,o bc l~rrwtit in wry lo\\. amounts. Its concctntration dwwwd probably dw to its hydrolJ?.sis to p(xptidrs 12-24 and 25-30. Aftw 2 mitt reaction timca, that awragc~ ttumbw of bonds ltydrol!*zcld ws O.XS. So IIWV pq~tidw wrc formc>d during this timcl but thaw wts a fut%hrr ittcrcaso in t81w :tmouttts of pcptidcs l-24 and 2-30. Pt~ptidc 17-30 dccrcawd in amount during this timcl compawd to tlw 1 min rc.action probabl!. as :I rwult of bGttg hydrolyzc>d to pcptidw 1724 and 2X30. Some>of tltc original oxidizcld I&cltuiti was still prcwnt. .iftor 5, 10 and 20 mitt hydrolysis 1.34, 1.70 and 135 bonds ww split, twpwtiwly, :tnd tlio clutioti pattcwls \verc qualitatiwly tltcl samcb (Figs. 1 and 2; 10 min data not slrontl). All of tltc> oxidized H-chain had dis:tppwwd and pcptidw l-24, O-16 and 25-30 I\-c’rc found in major amounts. Peptide 17-24, cbxpwtcid to b(bprcwttt, was not idclntifkd at tltcw timw but it ~vas undoubtrdly a comI)ott(wt of tltcl shouldw comprising fract)iotts z:! to G-1(,j mitt hvdrolvnis) as shown b\- the :ttt:Jysis at 120 &in (P’ig. 2). IXxpclcted p(p tid(t l-4 n-as not located in tlw analysis nlthough it, should lt:rw bwtt prcwttt in a lwrg(s amouni. I’cytidr l-3 was dr+ctcd at 80 mitt It~drol~ais. 13~ 60 mitt hydrolysis timcx (data not slton-11) tltc> amount of pc‘ptid(h l-24 had d(bcreawd cott.Gd(,rably and it had disappcnrctd at 120 mitt. At 60 mitt poptid(> l-3 \vas d(lt c~ctabl(k :tlottg \vith a mixture of uttidcntifkd pc’ptidw (pwk 5, 120 mitt). f+ptidc l-3 cont itiwd to iticrcwc in amount with furthc>r incubation timcl and rc~prcwntc~d ow of the thrw major p(>ptidrs prcwnt at 1440 and #SO mitt hydrolysis. .2ftw 2SSOminutes ItydrolJ-sic, 9 pc’ptid(xs \+yt-(’ clwrly d&wmitwd in the Itydrolyzatcb. I’cptidw l-3, .i-16 and Z-30 ww prwcnt in major amounts \I-ltik peptidcs .i-lT,, 17-21, 12-24, S-11, 25-30 and 21-24 \vCw prcwtit in

AN11 (1ALI)WI~:I,I,

minor amounts. Two pcytid(bs at t,ltcLb(yqitttting of th(> c~lution \wro not, idwtificd I’qtid(b 5-1.5 \V:W formcld by hydrolysis of’ t11(~ Lcu1S-Tyr16 bond probably of pclptidca T,-lfi. T’cpt,id(l ,5-11 \v:ts formed from It~~drolysis of t,hc L~u~~-V:tl~~bond prwumab~y of pqttid(l 516 and ~vas probably also p’cwrrt at 1440 mitt. l’cptid(, 2630 ws formed by hydrolysis of the> l’lt(~2S-Tyr26 bond of pqtidca 25-40. Through 120 mitt hydrolysis, pc~ptid(~2.5-30 US prcwttt in ma,jor amount but at 1340 mitt and 2SSO mitt it had Iargc~l>.butt cottwrtcd to pcytidcx WXO. l+ptid(~ “l-24 \Y:W formtld by splitting of tlw GIy2,1-C~lrt21 bond of poptid(> 17-24 and/or 12-24. In addition to the nittcl tl~~t~orrnittc~dpqtid(+ at 2SSO mitt OIN~ nould clxpwt tltat, pclptidw 1-4, 12-15, 12-16, 12-20 and 17-20 as ~(~11as tlw ftw amino acids t~wsitw, pltPtiylalattitte and glutamittc~ \vould b(l produced by c?xt,cwsiw actiott of E. parasitica protwsc~. Contamination by vxcws t~w)sitw and phwylalattittcl UX:: found in a ttumbw of casts but WV did not locatch thcl othc,r fiw c~xpc~ctcd p(lptidw. It is of itttcwst tltat IS ninhydrirtpositiw spots \v(w dctc&ckd aftclr high volt ag(l c~l(~ctropliorc,sis-i)~~p(,r cltrom:ltoi?;r:lpliic wparutiott of :I digst of oxidized fMtaitt (3). Four of tltcl spots \v(*r(’ prcwnt in trace amounts \~ltil(~ t~lirw ot~lic~r spots \v(w in small amourits. The protww

purifirld from h’ndothia paraas ckarly sho\~n b>the rclatiw ratw of hydrolysis of the suscc~ptibk peptido bonds of thcl oxidizcbd 13.chain of insulin. Tlw most susccyt8ibk bond in the: oxidizc>d B-chain to hydrolysis by Enclothia parasitica protww is tltc> I’hc~rr-l’hc~25bond. After otil> -0.25 mitt uttdw tltcs rwctiott conditions uwd t,his bond ~vas ltydrolyzcd in approximntc>ly 2.5‘;r, of the substrat(l mol(aculw. Bottds involving Tyr16-Lwl; and LoutsVal12 \VP~C also hydrolyzc~d initially to :I limitckd c>xtchtttin tltcl intact oxidizcld B-chain. Aiftc)r 1 mitt of wnction furthw It>-drolyix of tltc’ LcytII-Vai~~ bond bccamcl ittsigtttficattt~ and the> main points of hydrolysis ittcludtbd the Gin-Hiss, T~t.t~-L(~uli :md I’ltc~~-l’h(~~~ bonds as sho\~n by accumulation of pcptidw l-24, S-10 and 25-30 in major amounts. It appcwa that thcl TyrI,i-lJc~uli bond is mow sitica is an cwdoprotwsc

rwistant8 in thcx pclptidc l-24 than in thcx I,-I)lic~ri~l:~IaniIi:i~i~icl(~, b(~nz!-lox~curboll~~l-~,~~l~~~r~~lalxn~l-r,-pl~~~~~~lalanir~~~ and bcnzylintact oxidized B-chain since pcptidr l-24 ox~carbon;\-l-r,-~~l~~~~~~l~~l~~~~~l-~-~~~~~~~~l~~l~~~~~~~was found in major amounts evrn at 60 min. :imid(l :w not h>.drol\-zed by EndoUr in The> L~u~~-Vnl~~bond is particularly rwistant parasitica protcw;c~ (vwi \vith clnzymc’ conto hydrolysis in the> pq~tidc 1-lti as pcytidrl l-16 was tBhemajor pcytid(l found w(‘n aftclr ccntrations 100.fold largw tlwn used in this 2880 min reaction tinw and yptidc 5-11 MXS study :md . aftw 21 llr incubation (‘II)). This prwcwt’ in onI)* small amounts. The> caz\mc~ \\-ould utdlcatc~ tll:lt otlwr amino acid rwican only slowly II\-drolyzc tllc: L(w1S-Tyr16 durs OII OIW or both sidw of tllv P11(~zl-l’h(~2a bond to give pcytido 5-G. Thaw \VUSno (xvi- bond must b(> important in making this :\, partictul:u~ly suscoptibl(~ bond in thcl oxidizc4 d~ux~ of a pcptid(, bklg formed from a split of this bond in intact, oxidized B-ch:kt nor IS-chailr. ICatos of hydrolysis by othc>r protcw in tlw Ilydrolysis of pq)tidc l-2-1. l’cytidc Iytic cwzymcls II:I~VC~ bwn shon-n t’o be influcwwd by amino acid rwidwh not involwd ill :5-l;, ~1s prcwnt only in lo\\- amounts tww :Lft(tr ‘MO min of reaction. The, c~nz?;rw can formntion of thcb sus;cq)tibl(~ pqjtidc bond ;dso rc~mow I.‘hc,n~,lalanin(, from tlicl ,V-tcir- (30MVj). For (~x:lmpI~, rcwniir rapidly cl~~:~vc~s minal cwd of p(~ptldc~2,>-20 by splitting thcx :t l’llcs-:\I(+ bond itI A-c:will (36) ; I~OUYV~Y, l’h~~ss-Tyre6 bond bl,i ii dew so at :I VCYJ’ it is not able to I~~~tlrolyzc~this bond in tllcx slow r:1tct. pq~titlc b(~llz~los?c:lrbo,l~.l His-l’hc~(SOZ)Bonds involving (k-His5 and Tyr16- Lou17 .\Irt-C);\Ir (3.5). Th Iwptid(s bonds in tlrcs must b(’ hydrolyzcld at about thcx same’ ratcti; compounds L-l(~~,c\-1-r,-l(,ucirl:lmid(~ :md I,sinw no widww \vas found for accumulaIouc~I-L-phc~~~\-lal:~~~i~~:rmid~~ :w hydrolyzcbd tion of pc,ptidcs l-16 and 5-X. Thcl data of by k&r;thia, parusifica protcxw at a slo~v rat{’ Riclwrt (14) indicate that pc>pt8idcl-16 chrobut \vith :tbnorm:~l r:tto kirlc+ics (29). \I’(> :lr(’ mntograplis at the> s;amc: posit’iori :w tlrtx in tlicb procws ol’ si\~iithwizing various pqoxidized E-chain of insulin undw our c~xpc~r- tidw having thcx s~~~wu~~ arotmd tlw I’~P~,imwtal conditions. Ho\vwor, our data ind1%~~~bond of t11v osidizc4 Ikhnin to d(+clrctttc$ tluLt tlw pwk containing thca intact miiw \\-li~rv tliv major spwificit>- of’ tlrcb (7. oxidizc,d E-chaiil (-0.25 to 2 min) dow not z\-rncs is tliwctc~d. contain signifiwnt wnounts of pcytidcl l-16. In a st-ud!. of tllo :tctiori of a-chyniotrypsiit ls?r.rJothiaparasitica. protww has primary on insulill, Ginsburg :mtl Sclu~chman (37) spwificity for p+d(x bonds of oxidizc>d H- siiggwtt~d that once an ilisulin molwulo is chain of insulin involving amino acid rwiduc+ :ttkJwd by a-chymotrypsin t,hct insulin molwith nwtral alipllatic or aromatic sitl(b c~A0 is dqg:ulod to it 9 filial pc>ptidc fragch:tiriw. I’crliq3 of qua1 importxnw is tIi(l mtwts;. On tlic> 0tlicLr II:uK~, in an (kga.nt nwd for such rwiduw on c~ithw sid(l of tll(x study (10) on t I)(> rat<‘ of Ir>.drolysis of the susccytiblr bond as sho\vrl by splitting of thch oxidiwd H-chain of insulilr by trypsin n-hicll 1’11(~:!1-1’11(~2g T\.rl~-T~~~ulir L~~~ll-V:~ly~, GlnIlydrol~.zw tllcl A1rgZ&I\-2:I and LJX~~-:~~;\:~~ Hiss, and Asn$-Crl~l~ bonds. ,At pH 3.0, tlrcx bollds, it uxs ~l~o~~-rrtllat. tlw rwction folhistiditw of tlw Gin ,-His:, bond is positiwl\~ lo\\-(~1 t\vo I):tt Ir\v:iJ.s, w31 indcpcnd~wtl~ chnrg~d. Thcl bonds of LcwG-C\-stcki, Valr2- iilvolvirig tlicl ortlw of splitting of th(l two \7ally-Cystc~ic1y, c: ly2:,- pq)tid(l bonds. 111tllcs pr(w~~t \vorl;, the>data crl&I, Ala~,-I,!w~~, l’llf~:! / and TyrZ6-ThrZi I\-hich involw only- ;w(’ conclusivc~ tllat :i subhtr:it(’ molwul(~ is OIW:mino acid rwiduc~ with :I sizabk ncutrnl 11ot dcgrad(d to final protl~~+ once it 11:w alipliutic or aromatic side chain :w not hWI1 at taclwd. hydrolyzed. A not:tbl(s cbxccytion t,o this gy11cwlizatioll is tIw wry slon- Ii?-drol!-siy of the G1y2,,J;lurl bond found at 2SSO min. 011 tlw otlwr Iwld, bonds involving I’l~cyV&, \T:& AsII,~, Hiss-Lwti, H~s~,~-IAY,~~ :nl(l L~w~~-\~;~~~~ ;w not Ilydrolyzc~d.

60

WILLIAMS. TABLE

WHITAKI:lI,

III

HYDROLYTIC

ACTION OF SF:VI;.R.~L PROTEOLYTIC B-CHAIN ENZYMES ON THE OXIDIZED

OF INSI,-IAN Bond hydrolyzed Phe,

-

Valz

Asna GIna Leull

-

Gin, His:, Val,,

Glula Ala 14 Len16 -

Ala14 Leuli, TyrlG

TyrlG

Leul,

-

Leu 17- Vall8 Argzz - G~SZS Gly,, - Glua Gly 23 - Phepa Phe,c - Pheza Phezj -

Tyr26

TyrQB LYSS -

Thr?, Alar0

.4X1> CAI,l)WRI,T,

gcncral specificity may bcbsimilar to t’hat, of pepsin, it. difY(w from lwpsin in it,s dctail(~cl specificity. This has bwn rqw%cd to b(l trw of other acid protoasc’s (:35).

Enzymes exhibiting actiona pepb, rent, papd, thyme, m.p.‘, m.m.g e.p.h Pep, C.P. pep, ren, pap, chum, m.p., e.p. PCP, ren, pap, chum pep, re11, pap, m.e. pep, ren, pap, chum, m.p., m.m., e.p. pee, ren, pap, chum, m.p., mm., e.p. ren, pap, m.p. tryh e-p. pep pep, ren, chym, m.p., m.m., e.p. pep, ren, chym, m.p., m.m., e.p. chym try

a Abbreviations are: pep, pepsin; ren, rennin; pap, papain; chym, chymotrypsin; m.p., ic1ucor pusillus protease; m.m., Mucor miehei protease; try, trypsin; e.p., Endothin parasitica protease. b Ref. 7. c Ref. 12. dRef. 10. e Ref. 38. f Ref. 39. QRef. 14. h Present work.

oxidized B-chain of insulin hydrolyzed by several enzymes arc shown in Table III for comparison. Endothia parasitica protease hydrolyzes bonds involving GlnJ-Hiss, LCUW Vallz, LeulS-Tyr16, Tyr1G-L(w17, Phcz*-I’hczb and Phe25-Tyr26 in common with pepsin but, fails t.o hydrolyze bonds involving Phel-Va12, Glula-Alall, AlaIr-Leuls and G1~3-Phe2.~which arc hydrolyzed by pepsin. On the other hand, Endothiu parasitica pro&w hydrolyzes bonds involving Asnz-GlnA and Gly~Glunl not hydrolyzed by pepsin. Endothia parasit&a prokase does not hydrolyze the typical pepsin subst,rate bwzylox\rcarbonyl-L-glutamyl-r,-tyrosine (3) indicating t,hat, while its

1. HIGEMEYER, K., F.kww.~,, I., .\ND WHIT,~KER, .J. R., J. Dairy Sci. 61, 1916 (1!)68). J. L., Appl. Microbial. 16, 248 2. %\RDIN:~S, (1968). J. R., 1. 3. LARSON, 11. K., .lsD WIIITXER, Dairy Sci. 53, 253 (1970). M. K., .~ND WHIT~ICETL, J. Ii.., .J. 4. L.us~x, Dairy Sci. 63, 2G2 (1970). J. R., JJelhodn Enzymol. 19, 436 5. WHIT.~KER, (1970). F., .SNU TUPI~Y, H., Ninchem. .I. 49, G. SXGER, 481 (1951). J. T., OTTESXN, M., SVENDSEN, I., 7. JOK\NSEN, .\ND WYBIC.\NDT, G., Compt. Rend. Trnv. Lab. Carlsberg 36, 365 (1968). 8. NAUGIITON, M. A., AND S~XGER, F., Hiochem. J. 70, 41’ (1958). A. s., AND h\V.lR, Ii. A., Bio9. NAR.\YANAN, chenr. J. 114, 11 (19G9). J. T., :\XD OTTESEN, P*I., C. Iz. 10. JOH,~NSEN, Tmv. Lab. Curlsberg 36, 265 (1968). Y., Biochim. 11. EHhTh, M., \SD TX.\HISHI, Biophys. Actu 118, 201 (1966). FOLTK\NN, B., AND 12. J%AX~-JESSJSN, V., RO.XI3‘1UTS , W., Compt. Rend. Trav. Lab. Cadsberg 34, 326 (1964). A. P., LXcLEltC:, J., .\ND F~LLA, F., 13. RYLE, Biochem. J. 110, 4P (1968). W., C,‘. K. Trw. Lab. Carlsberg 38, 14. RICKEILT, 1 (1970). B. I., STEIN, W. H., Xl~~ MOORE, S., 15. Gmtwx, J. Biol. Chem. 241, 3331 (1966). lti. GIXOSKOIBF, W. R., HSIEH, B., SUMMSRI.~, L., AND ROI~ISS, K. C., Biochim. Biophys. Ada 168, 376 (1968). II., S.&%I, R., SINGER, A., AND 15. MATSUll.\lt.& 116, JUICES, T. II., Arch. Biochem. Biophys. 324 (1966). 18. FEDFXL, J., .~SD LE\V.IS, C., .JR., Biochem’ Biophlys. Res. Commun. 28, 318 (1967). 19. WANG, S. S., ions C~RIBENTER, F. H., Biochemistry 6, 215 (1967). I,. W., AND BROTVN, C. S., J. 20. CUNNINGII~Y~I, Biol. Chem. 221, 287 (1956). H., &O21. ~~r1~l~IOND, B. R., ASD GUTFREUNI), chem. J. 61, 187 (1955). R. B., ;~ND NIEMBNN, C., J. Amer. 22. M1~wrN, Chern. Sot. 80, 1481 (1958); WOLF, J. P., .ZND NIElVSN, C., .J. Amer. Ch.em.. Sot. 81, 1012 (1959).

E. PARASITICA

PROTEASE

ACTION

23. CRAIG, L. C., KONIGSBEKG, W., L4~~ KIP~G, T. I’., Biochem. Prep. 8, 70 (1961). 24. MOORE, S., AND STEIN, W. H., J. Hiol. Chem. 211, 907 (1954). 25. HanEEf!, A. F. S. A., Anal. Biochem. 14, 328 (1966). 26. Moorw., S., AXD STEIN! W. H., J. Bid. Chem. 211, 893 (1954). 27. SPACICMANN,D.H., STEIN, W.H.,asr, MOORE, S., Anal. Chem. 30, 1190 (1958). 28. MOOEE, S., AND STEIN, W. H., Blefhodn Enzymol. 6, 819 (1963). 29. WHITTAKER,J. R.., unpublished data, 1971. 30. SCHECHTER,~.,Ani~an~owI!cz,N., AND BERGER, A., Ier. J. Chem. 3, 101 (1966). 21. SCHECHTER,I., .~XD BERGER, A., Biochemistry 6, 3371 (1966).

ON OXIDIZED

B-CHAIN

OF INSULIN

61

32. SCHXCIITEH, I.? AXD BEKGER, .4., Biochem. Riophys. Res. Commun. $7, 157 (1967). 33. MEDZIHILIDSZKY, K., VOI-NICI;, I. M., ?~/~EDZIHR.~DS~I;Y-SCH\\.EIGE~~,H., AND FRUTON, J. S., Biochemistry 9, 1154 (1970). R. C., L~~~BLOUT, E., Proc. Naf. 34. TrronimoN, dcad. Ski. 7,y.S.i4. 67, 1734 (1970). 35. VOYNICIC,I.?bI., .IND FRUTON, J. S., Proc. Kai. zlcad. Sci. I:.S.A. 68, 257 (19il). 36. JOLLES, J., JOLLES, I'., UD ALMS, C., N&w-e London 222, 668 (1969). 37. GINSIIERG, A., -LSD SCHXHLIN. H. K., J. Bid. Chem. 236, 108 (1960). 38. DESNUELLE, I'., Enzpes 4, 93 (1959). 39. MCCULLOUGH, J. RI., .~ND WIIITXKER, J. R., J. Dairiy Sci., 64, 1575 (1971).