IN V ESTIG A TIO N OF PROTEINASE PROTEIN IN H IBITO RS (PPIs) FRO M SEEDS OF CERTAIN PLANTS BELONGING

VNU. JOURNAL O F SCIENCE. N at. Set.,

t.xv,

n^3 - 1999

IN V ESTIG A TIO N OF PROTEINASE PROTEIN IN H IBITO RS (PPIs) FRO M SEEDS OF CERTAIN PLANTS BELONGING

TO M ORACEAE FAMILY AN D OTHER ONES*

P h a m T ra n C h a u , N g u y e n H o n g A n h , Le T ro n g Q u a n g P h a n T h i H a , N g u y e n T u y e t M a i, D ao T ro n g A n h .

Biotechnology Center, College o f Nãtìiraì Sciences - VNU

I. INTRODUCTION

Proteinase protein inhibitors (PPIs) plav an important role in living system aiid have several practical applications [7, 9, 11]. Therefore, the investigation of PP Is ill plants lias both scientific and roalistic significance.

However, ill Victiiaiii, the study on PPIs has started in our laboratory only since 1986. During th a t period, most of studies were focused on PPIs from Cucurbitaceae seeds.

As far as wo know, there arc luoro than 90 published papers about protein from Artocarpus seeds, b u t no one reported about PPIs.

This work presents some prcliuiiiiarv results of PPIs from seeds of ccrtaiu Moraccac plants and from some plants of other faiuilios.

II. MATERIALS AND METHODS 1. M a te ria ls

T i u ‘ o f A i to c u i ]}UƠ i t i c l u i o j . y l a G u g li, Árioctn-pXLO c ĩ i a p ỉ a s h a RoxV>; A v t o r n r -

pus intei'ger ; Artocai'jms lakoocha Roxb; Steblus asperLoni; Annona squamosa L;

Diospyjvs kaki Linn f; Abeimoschus esculentns L; Syzygmm, jambos L. ; Delavaya toxo- cai'pa; Ciừ^ts gm ndis Osbock; Cifnts sinensis L; Pynts pyTi-folia Nakai; CaHca papaya L;

Sechixmi edule Sw; Clirụsophyllmìì cainito L; Persivim.on kahi soft type ripe were colloctccl fioiu Iiiatme fruits.

' (-h) Chemicals

a - chyiuotrypsiii from Lab Lciirquiii (France); casciu from BDH clioiuicalH Ltd, Scpliadox - G 75 from Phanuacia Fine chemical (Sweden); tr\psiii, albuiiliu wore supplied by Sigiiia Ltd (USA).

*) This work was financially sponsored by ” Basic scientific program m e - project", subproject 6.4.4.

A b b re v ia tio n s : PA: proteolytic activity; TIA; trypsin inhibitor activity; ChIA; chym otrypsin in­

hibitor activity; DChIA (D TIA ): Tl or Chi peak.

- Chemicals for electrophoresis: acrylaniide, bis - acrylaiiiidc, Tris - basc...ctc., from Sigina Ltd (USA). The other chemicals reached the PA giade.

(-h) Methods

The finely ground seeds were extracted with distilled w ater (with ail exception for Diospyros kaki Linn seeds, where pH of 4.5 Britton and Robinson buffer was used).

The ớbtained suspensions were stirred for 30 minutes at room tem perature aiid claiified by centrifugation (8000 rpm) for 10 miuutes. The supem ataiit was collected for further analysis.

- Protein concentration was determined by the Lowry m ethod [6], using bovine serum albumin as staiidiqrd.

- Determination of proteolytic activity (PA) was carried out by Ansou modified m ethod [8]. PA was determined at 35.5^c using pH 7.6 Srrensen buffer with 1% casein as substrate for 20 minutes. One unit of proteolytic activity was defined as the amount of enzyme after one Iiiinute of action at 35.5°c generating TCA soluble products, giving colour reaction with Folin Ciocalteau reagent equivalent to one micromole of tyrosiiie.

Inhibitory activity was deteniiiued from the residual enzyiuatic activity after 10 mill pre-iucubation of the Guzyiue with the iiứiibitoi solution. Oue unit of inhibitorv activity was defiued as th at anioimt of inhibitor, which reduces the activity of two mg of eiizyiiie by 50%.

- PA, ChIA, TIA were also detenninểd by diffusion m ethod [5,10 .

Heat stability of TI and Chi was estimated as follows: the cnidc extract was treated at lOO^C for 15 luiii, cooling iimnediately in icc. The activity was measured in standard conditions. The remainiug activity was calculated in coiripariiig with the initial activity.

- Polyaciylaiiiido gel electrophoresis for identification of protein bands was pci- fomicd according to Laemiiili method [4]. Protein pattern was analysed by Sliaip JX. 330 scaiiiior.

Gel Ịìlừution by Sephadex~G75 cohirrin

The gel filtration cohiimi witli a size of 1.7 X 92cni was equilibrated by pH 6.5 Srreuscu buffer for AHocarpus melinoxyla Gagii and AHocarpus chaplasha Roxb pH 7.6 for Abelm.oschus esculentiLS L. aiid Delavaya toxocarpa. Protoiiis were ohitcd by the equi­

librated buffer. The fraction volume was 3ml. The niiiiiiiig speed was 20inl/houi.

III. RESULTS

1- S c re e n in g p ro te in a s e in h ib ito ry a c tiv ity (P IA ) o f s e e d c r u d e e x tr a c ts

Seventeen seed samples of Moraceacy Rutuceae, Annonaceae, Rosaceae, Ebenaceae, Caricaceacj Malvaceae, Sapindaceacj Myrtaceae, Sapotaceae, fam ilies were subjected to PIA investigation. Nine of them showed iiiliibitory activity (Table 1) blit in the right others: pomelo, orange, peai’, papaya, diayote, star apple, pcrsiiiunon, kaki soft ripe neither TI nor Clil wOvS discovered.

2 P ham Tran Chau, Nguyen Hong Anh, Le TYong Quang. . .

Table 1, 4. .

In vestig a tio n o f P ro te in a se P rotein Inhibitors (P P Is) . . .

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P ham Tran Chau, Nguyen Hong Anh, Le Trong Quang, As shown ill table 1, Artocai'pus nielinoxyla Gagii, Artoairpus chaplasha Roxb, A b e h w s c h u s excnlentĩLS L and DeẦavaya toxocarpa co n tain b o tli T IA a n d CliIA. However ill the first two samples. ChIA was 2-3 times higher tliaii TIA, while ill Abelmoschus escidenius L. and Delavaya toxocar'pa, there was no clear distinction between TIA and ChlA.

In 1 cniaincd samples, TIA was only discovered in seeds of Artocarpĩis lakoocha Roxb Dtospyros kaki Liliii f, Delavaya toxocai'pa, Annona squamosa L. while KIA existed in Artocarpĩís melinoxyla Gagii. Among the Moraccae soods owing PPI, the highest TIA lo\rl (5675 n iiu /g of dry uiattor) was foiiiid in Artocai'jms. Melinoxyla Gagii. In opposition Sfeblus aspei' Lour sam ple showed prot.oolytic a ctiv ity [ I J S l U / g of d ry Iiiatto r) blit, was lacking in PA, PPIs. Tims in comparison with the Moniordica seeds, TIA of Artocaiytis was 10 tiiiies lower [12, 13] but it was higher tliaii punipkiii, squash, w atrr melou... TIA.

Basing on this oxpcriuioiit, W(' .scloctcd 4 saiiiỊ)l(‘s of Al'tocarpas iiidinoiyyla Gagn;

ArtocaiyxLS chaplasha Roxb, Delavaya toxocarpa and Abelviochus escideiitus L. for fiuthoi analysis.

At first, SDS - PA G E was u sed for clctoctiiig th e p io to u l coinpoiiouts.

Number

Rm

Artocarpus melinoxyla Gagn

Artocarpus chaplasha

Abeim oschus esculentus L

Delavaya toxocarpa

1 0,14 + - + -

2 0,18 + - + -

3 0.2

4 0,21 + - + -

5 0,23 + - + -

6 0.24 +

Ị 0.2t) + - + - +

8 0,28 +

9 0.29 + - + -

10 0,32 + - + -

11 0,36 +++

12 0,39 + +++

13 0,44 ++ + +

14 0,45 +

15 0,50

16 0,54 +

17 0,58 ++ + -

18 0.62 +++ +++

19 0,70 +++

20 0,83 +

21 0,85 + -

22 0,90 +

Table 2: Tlio ralativc mobility (Riii) of protein bands from 4 samples.

In ve stig a tio n o f P roteinase P ro te in Inhibitors (P P Is) .

2. E le c tro p h o re s is p a t t e r n o f se e d e x tr a c t o f A r to c a r p u s m e lin o x y la G ag n ; A r to c a r p u s c h a p la s h a R o x b ; A b e lm o c h u s e s c u le n tu s L; D elav ay a to x o c a r p a L

For convenience, the Rin of protein bands was calculated and listed ill table 2.

The d ata in photo 1 showed th a t the protein pattern of seed extract of AHocaryus melinoxyla Gagii and Artocarptis chaplasha Roxb was rather similar, including 10 protein bauds in Artocarpus melinoTtjla Gagii and 11 ones in Artocarpus chaplasha Roxb. Among these bands, the protein ones with Rin=0.39;

0.44; 0,58; and 0.62 were inajor bands.

It was iiiterestiug to note that there was only one difference ill protein band with Rju=:0.39 between Artocarpus vielinoxyla Gagii

a n d A r fo c a r p u s ch a p la sh a R o x b . M a y b o tlic

differentiation between Artocai'pus nielinoxyla Gagii and Artocarpus chaplasha Roxb was de­

fined by this protein.

The rate ratio of main protc'in bands (Rni = 0.39; 0.44; 0.58 and 0.62) was calcu­

lated and presoiitecl ill table 3. In Artocar-

jn i s m eiiT w :ry la G a g ii, tlio p r o t e in b a n d w it h PI 4 1 . • r I . .

' J liofo 1. piotriu iJattn n of ('Xtiact

Rill = ,0.39 a c c o u n ts foi a p p io x iia a t o ly 6. IG ^ ^ tr o iu A r t o c a i p i i s liK 'linoxyla G a g a ( 1, 3 )A *. I 1 - /1

of tlio to tal pioteiii coiitnit. 6.16 % whilo for , I A . 1 1 1 r. 1 /0

’ aiul A i t o c a i p u s cha])lasha Rd xI) (2, 4) tlio R n i = 0.4 4 p io t o iii h a ix l it w as

r>‘) % , ,, , , , , . ,

1, r iie li l a iif ('(Jilla m lu I I I ()1 sa iu {)l(-

lowt'i' Mian that of tlir coriPspoiKliiie, one ill .5, 4: ('Hcli laiK' coulaiii 1-)

r, ,

^{, , „ , • , r} ỊIÌ C)i saniplr^{; r ,} Artocarfius cliaplashaRoxh. Thus, a(iucstioii

arises; d o c s tlir p r o te in band w ith R m = 0 .3 9 o rigin ate ÍI0111 thí' one wi t h Hni ^ 0.44? aiul

the coriTspoiidiug goii(s) klmtifviiig the (lifỉ'('i('iic(' l)('l\v('('n t!i('s(« two Artixarfiis spades!

The major protein bands (Km) Samples '

0,39 0,44 0,58 0,62

Artocarpus chaplasha Roxb

(column2) ⁰⁹ 23,65 36,47

Artocarpus melinoxyla Gagn

(column 1) 6,16 18,6 24,65 29,39

Table 3: The porcoiitago ratio of luajoi proti'iii bauds ill c()iiiỊ)ai isoii with the total of dotcctod bauds bv Sharp JX-330 scaiuior.

The SDS - PAGE results also showed tlio difforeiicr hi clcctrophorosis pattoni aaoiig Artocai'pus ĩneẦỉnoxyla Gagii; AHocarpus chaplasha Roxb and Delavaya toxocarpa The shiiplost protein pattern with 5 protein bands ill which the major one with Riii = 0.Í6 was foiiiic] in Abelniocfms escĩdenÌMS L. Although, ton protein bauds were dctectcd ill p.'Otciii pattern of Delavaya toxocarpa, almost tliese proteins wore different from the concspciidiiig ones of the other families ill Riu value.

To preliminary separate PPIs from crude cxt,ract, Sephadex G75 coliuuii was used for chromatography.

3. C h ro m a to g ra m o f se e d e x tr a c t o f sa m p le s th r o u g h S e p h a d e x - G 75 colum n As shown in iig. 1 and fig. 2, the chroniatogxaiii patterns of Artocaryus rn,tli'ioaryla Gagii and Artocarpus chaplasha Roxb [5, 10] were siinilai' ill shape, consisting of 2 peaks (D i,D9), in which D \ was an active peak. Ill AHocarpiis vielinoxyla Gagii, D i naclied 52.78 % of the total protein loaded oil the coluiiiii while it W£LS 55.75 % in AHocarpxLS chaplasha Roxb. Thus, at the back of separation, the puriiiod level of Chi hi Artocarpus melinoxyki Gagii was equivalent to th a t of Artocarpus chaplasha Roxb. After chroiaatog- raphy, since TIA both of AHocarpiis melinoxyla Gagn, Artocarpus chaplasha Roxb was very low, it was not possible to dctcct by Anson uiotliod, TIA was only discoveiod by diffusion niothod.

It should be noted th at TIA fraction shared the same peak with CliIA OIIC- This pheiiomenoii may be the cause of the similarity ill Rill between TI and Chi in these samples? or there existed the double headed inhibitors ill Artocarpus: one doinaiii iiiiibits trvpsiii, the other is completed witli chyuiotrypsiii, following the typo of BBI fcrni in soybean seed [2, 3]?

Under the same couditiou, thcic wore SOUIO differences ill chroiuatogiaiu pattern of Delavaya toxocarpa and Abelmodms esơidentĩLS L. PPIs fractious were eluted slowlicr, thus it documoiitod th at luoloculai weight of PPI ili these samples was lower than that of A rtocarpĩis.

I"ig. 1 showed two main piotoin peaks {Dị, Do) ill chroiiiatograiu pattern of sood cxtract of Abelniock'us esculentus L.. TIA, CliIA wore shown ill D\ As the protein contciit of Di roachod 35.71 % of total studied protein, the specific activity of TIA and CliIA iiicrcased rrspcctivoly 3.15 and 3.35 tiiiios.

As shown ill fig. 1, there wore 3 peaks {Dị. D2 and Ơ3) in chroinatography pattern of Delavaya toxocarpa. Since D2 had the lowest protein coiitciit (about 26.5 %) and was a TI peak, thus the specific activity of TI increased 3 tiiiios more than that of initial extract.

4. E ffect o f lie a t tr e a tm e n t o n C h IA (o r T IA ) o f s tu d ie d sa m p le s

It is known that high PPI content ill footl might affect unfavorably digestion ill aiiiiiials and humans duo to the docrcasr of cnzyinc activity by inhibitors.

- .4.9 Artocarpiis vielinoxyla Gagii and Abebnochus escnlentus L. has been used as a good initiiont, wo studied the impact of heat troatiiiont at lOO^C for 15 luiuutos oil TI,

Chỉ in those saiiiplos.

6 Pham Tran Chau, Nguyen Hong Anh, Le Trong QuariỊ. . .

Fig. 1: Chromatogpaphy p attern of seed extract of studied samples by Sephadex G 75 column

a) A rtocarpus mekmoxyla Gagn c) Delavaya toxocarpa b) Ratocarpus chaplasha Roxb d Abelmochus esculentus In ve stig a tio n o f P rotein ase P ro te in Inhibitors (PPIa) .

Pf actions

(b)

12

10

ÍÍ ÍỈ s 5! s? s R

Fractions

f k M i % .

^ R S ? S 8 ỉ 5 8 8 8 g 8 Practions

(đ)

TIA KIA Piotein

Pham Tran Chau, Nguyen H ong Anh, Le Trong Quang,

Aíỉocarpus Arỉocâíípus Abdfnochus chđplâshd meiitioxyld eaciientưs L Roi Gốgn

C tixle exffact

D ela v ay a A tto c a ip u s Aiỉocafp*,Ji A tfcca iip u s

ỉoxocàĩpâ chaplasha mdmũxylâ fr.ei*no5<ylô

R o x b G ag n G d g n

Active peak (CWA 0{ TiA) oòỉ^-ied Sepha«jex chforn^ogidphy

Fig.2: The cffcct of heat treatiueiit on CliIA (or TIA) of studied samples.

- The obtained results showed th at when the seed extract was treated at lOO^C for 15 iniuutes, TIA arid CblA of Abelmochus esculentiis L. were almost destro^tĩd. Ill

Artocarpus melinoxyla Gagli, Artocarpus chaplasha Roxb, CliIA ill both cnidc extract and active peak (DchiA,-Dtia) were reduced by aroiilid 70 % but the remained TIA of seed extract ill these samples was still high. This phenomenon might be due to the fact th a t sample concentration for deteniiiiiiiig TIA was 5 times higher than th at one for Clil detection. Therefore, TIA, ChIA ill boiled seeds were detcimined in a further step.

After boiling, TIA and CliIA rcdiiced to 10 tiiues ill coiuparisoii with initial activity, correspoudiug to 0.1 - 0.2 lU. The ininiuuuu of trvpsiii coiitcut released from hiiiiian pancreas per clay is 782 irig (782 Ư) [1], thorcfoio the roiuainhig TI, Chi in AHocaTpiLS seed after boiling has no considerable iinpact on trvpsin and chvinotrvsin activities in liuiiiaii body.

IV. CONCLUSION

1- Among 17 iiivostigatod samples, TI aiid Clil are found in 9 samples, consisting of ArtocaTpus w.elino:ujla Gagii, . Artocarpus chaplasha Roxb; Artocaiyus integer Moir;

tocarpĩis lakoocha Roxb; Streblus asper Lour; Annona sqttaniosa L; Dispyros kaki. Linn f;

Diospyros kaki. I ll which 4 samples of ArfocaiyiLs nielinoxyla Gagii, ArtocarpĩLS chaplasha Roxb Syzygiiiin jauibos L., Abchnosclms escuiciitus L., had both TI aiid Chi.

2- pH 8,3 SDS - PAGE 12,5 % has enabled the idcutificatioli of 10 protein bands from seed extract of Artocarjms ĩìieỉinoxyla Gagn, . Artocai'pvs chaplasha Roxb. However, there was another protein band with Riu = 0.39 iu Artocarpus melinoxyla Gagn, at the saiuc time, the protoin with Riii — 0.44 was decreased.

At least, 5 protoin bands were found ill Abehwchus escĩãentus L. and 9 protein ones were discovered ill Delavaya toxocaiya. Almost those baiids difforod ill tenus of Rrn value to the coiTOspondiiig other samples.

3- Chiomatograni pattoni of AHocaiyus nielinoxyla Gagii; Artocaiyus chaplasha Roxb

wore rathoi siuular, consisting of 2 peaks (D i, D ,) hi which D , had both TIA aiul ChlA.

- I I and Chi of seed cxtiact of Artocarpus Tri.elinoxyla Gagn; Artocarpus chaplasha were After heat treatm ent at 100°c for 15 m im Z s the K'liiaiiK'tl activity was over 30%.

V. REFERENCES

1] R.M. Bonio , M. N. N. Levy. Physiology , Mosby - Yoai' Book, Inc., USA (1993)669-

67G. ’

[2] Y. Bilk , A. Gorier. Proc Int. Res. Conf. Inhibitor., Int. ed. H. fritz. H. Tes chesche 142-1Ị8. Berlin; De Gruftcr (1971)304.

[3] D.E Bowman. Diffcrcutiatioii of Soybean antitryptic factors, Proc. Soc Exp Biol.

Med. 63(1946) 547-550.

4] V.K. Laeiiuiili. Cleavage of stnictm o protein during the assoiiibly of the lioad of bactcrophagc T4, Nature 227(1970) 680-685.

5] Lohik Jacok, Pham Till Trail Chau, Kicloczawa Jacck. Zastosowanio edcstviiy do vadaiiia aktywiio’sc’ proteiiiaz i icli inhibitorow, X X I Zjazd P.T.Bioch. K m L Z Streszczenie 140(1985).

|6| Q H^Lowry, N.J^ Rose b.cugl,, AX. Fan , R.J^ Rai.dall. p.otain with tlio Foliii phenol reagent, J. Biol. Chem.. 193(1951) 265-270.

|7] Ng,,v<-„ Khoa Dion Tl,„, PI,a,,, Tl.i Tia.. Cl,an, Prclimmary roscarchcs on tl.o s'-paiatii.g from Mo.uodica seed to R a t i reproduction, Journal o f Biologs-Soil science: Hanoi National University (1989)

w M.M

W incjunajtc. OpreJelcute piotcolitichcskoi aktivuosti k n m M - Iiykh prcparatov niikrobiologichoskovo proiskliachdenia, Pnklad. Biochern. Mikro-

bUu. 2 (1 9 0 0 ) 232.

Chau. TVi/psm inhiUtors of white bush (CucnrbUa pepo var, patisso- nia) jn n t anh seeds. (Doctor babilitatus thesis), Acta Uiiivorsitatis Wratislavion^s (198o) 912.

[10] Pham Thi Tran Chan, L. Koiiopska, J. Lehik. TiyiJsin inhibitors ill tlio alouroiio gian io so fC u cu rb itap ep o v ai. patissonia (White bush) cotylodous, Brochem. Pi>z.

*o/., paaiizoii, 181(1986) 565-569.

[11] Phani Tin Tran Chan, Nguyou Tiiyct Mai, Phan Thi Ha. Purification, cliaractoriza- tioii of autilysin fioin bovine Imig - preliminary study regarding the detonuhiatiou ot Its practical uses, Journal o f Biology 15(4)(1993) 8-10 (ill Viotiiamcs).

,12] Pham Thi Tian Chau, V.T.Hion, P.T.Ha, N.H.M.Q„yon, N.T.Mai. Proliniiiiary data of trypsin inhibitors (Tls) from squash seeds of Virtnaui, 10th FAOB syvipo- sruvi on protein Research 7-10/12/1993

13| PUa„ T„a„ Nghia, Pl.au, Thi T,a.. Cl.ãũ (1987): a Z g c s to trvpsh. i„l,ibito.y ac- t vitv (TI A) aiKl proteolytic activity of develop,uct seeds ami fii.its of Momoulica chaiaiitia L, Jowmal of Biology 9(3), 12-17 (ill Victuaiiios).

In vestigation o f P roteinase Prxitein Inhibitors (P P Is) g

TAP CHÍ KHOA HOC ĐHQGHN. KHTN, t.x v . n°3 - 1999

ĐIỀƯ TRA CÁC PROTEIN ỨC CHỄ PROTEINAZ (PPI)

o HẠT MỘT SÓ CÂY THƯỘC HỌ MORACEAE VÀ MỘT VÀI HỌ KHÁC.

P h a m T râ n C h â u , N g u y ễ n H ồ n g A n h , Lê T ro n g Q u a n g P h a n T h ị H à , N g u y ễ n T u y ế t M a i, Đ ào T rọ n g A n h

7>uíjg Tàin Công nghệ Sinh bọc Đại học K H T ụ nhiên - ĐHQG Hầ Nộí

Chúng tôi đã tiếu hàiih điều tra sa bộ PPI của 17 mẫu hạt thuộc các họ Dâii tằiu, Bỏng, Siiu, Bồ hòn, Tliị và Na. Tioiig đó 4 uiẫu hạt Mít rnật, Mít dai, Roi, Đậu bắp có chứa cả chất ức chế tripxiii và kiinotripxiu. Năiu niẫu hạt khác: Chay, Mít tố nữ, Hồiig đỏ, Na có chứa TI hoặc KI. Tír các kết quả trên cúng tôi đà cliọii hạt Mít luật và Mít dai để nghiên cứu tiếp. Kết quả điệu di dịch chiết hạt 2 Iiiẫiỉ Mít m ật và Mít dai cho thấy pliổ điệii di proteiii 2 mẫu Iiày khá giống uhau, trừ băiig protein có Riu = 0.39 (Mít dai kliỏiig có băug proteiii uày). Phải cliăiig băng proteiii trên qưi định Hự sai khác giữa Mít m ật và Mít dai. sắc ký qua cột Sephadex G75 cũiig cho thấy phổ sắc kí 2 m ẫu này khá giống nhau gồm 2 đỉiih protein chính, troug đó đỉnh tliứ Iihất có cả KIA và TIA uhưng ờ mức độ th ấp liơii. TI và KI từ hạt luít khá bều vói uliiột, sau khi sử Iv ờ 100'^c trong

15 phút, hoạt độ kìm hãin chỉ còn lại khoảng 30 % so với hoạt độ ban đầu.

10 P ham Tran ChaUf Nguyen Hong Anh, Le Trong Quang, . .