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JPRS : 17p239

ARTICLES PROM, L1AD P AEX1IIN&UK(BIOPHYS105)

-USSR-

PFollowing is the translation of two articlesn the Russian-language periodical Dklady

A45demU Na (Reports of the Acadern6f -SciencesUSSR)., No 4, Moscow, 1962. Additional tbliogra*iieInformation ae&ampanies eadh article. J

Contents

Protective Effect of Phytolipopolysaocharidesand VB-2 AgainstIrradiation...... * ... ..*............

On the Selective Effect of Ultrasonic Waves onthe Molecular Structure of Insulin..............•. 7

a

130f0V In= OF MWTOC O LSAOSAUMJ AND M,-2

fe.tus.ag .is the tnamaia of anm articole bTo A* fmd*, Ue.P. G1'rsa,.U Be No Gladysher,"mt YV Z. tuslikov in the o.eibasMme

I~i~e (R WONALMA of theA~ui of Be~me N , 4osow. 1962,

(Presented by Academician A.I. Oparin 13 April 1962)

We know that the bacterial lipopolysacoharides (BLP)have a pronounced effect on the functioning of human andanimal bone marrow. This Is manifested in heightened leuco-cytosis (with an intensification of the phagocytio activityof the leucocytes and reticulo-endothelial cells), whichapparently strengthens the resistance of man and animals toinfection (the BLPs do not affect erythro-thrombopoiesis)(refs 1-9). However, the high toxicity of the BLPs and theirability to stimulate fibrinolysis hinder the use of thesecompounds as protective agents against penetrating radiation.

Por this reason, from the standpoint of research ondefense against penetrating radiation, the compounds of thelipopolysaooharide type found by B.N. Gladyshev (BiochemistryInstitute of the Academy of Sciences USSR) in the higherplants (phytolipopolysacoharides -- PLP) (refs 10, 11) couldbe of some interest. The existqnoe of a ohemostructuralsimilarity between the BLPs and PLPs led to the suppositionthat the PIPs might also exhibit biological activity, includ-Ing that of stimulating in some degree the functioning ofbone marrow. We also surmised, bearing in mind the origin of-the PLPxs that the latter lack or possess very low toxicity.

The present study, a continuation of the search forprotective agents against penetrating radiation (ref 12),involved tests of a preparation made from tea leaves whichdoes not exhibit a fibrinolytic effect (oral presentationby G.V. Andreyenko, Moscow University). The intestinalmucosa was protected from radiation (ref's 13, 14) and exhaus-tive diarrhea prevented by means of the VD-2 prepartionwhich is a vinylbutyl ether polymer (mol wt 6000; n•0 1 .4600;viscosity in 104 benzene solution 6-10 centistokes).

w1thed The study was carried out on *hit* male ratewith no I nomograms, Both the test, and control specimenswere subjeoted to total irradiation with Y-rays from a

0060 source (700 r does) under the following technical con-ditions: DSM-49, dose rate 62 r/min. For uniform Y -irradia-tion, the rats were treated in groups of 5-10. The animalsin group I received four suboutaneous injections of PLP eachin the pelvio area for a total of: 200-400 per rat weighing200 grams, with each injection oontAining 100-50( 'of sterilesolution of the agent (sterilization in autoolave): let *injection 96 hours, 2nd inSeotion 72 hours, 3rd -- 48 hours,4th -- 24 hours prior to irradiation. The specimens ingroup I1 were tested for combined effect of PLP an .VB-2preparation: the latter was administered perorally 24 hoursafter irradiation and then daily throughout the entire ob-servation period (30 days). The animals in group ITI wereused for control. Peripheral blood was studied for all animals.Determinations were made of the hemoglobin, erythrocytes, .leuoooytes and leuooo te formula, retioulooytes, and thrombo-eytes., The state of te blood was studied prior to Irradia-tion and after various time intervals (1; 5; 10; 15e 20 and30 days) upon irradiation. The data were processed statis-tically by V.I. Suslov on the X-2 computer.

1" Rosultg. The study of the proteotive effects of thePLP knd V34 15teparation revealed that with the administra-tion of the PLP preparation 24 hours in advance of irradiation,the death rate among the animals was 26%. With combined pro-teotion the rate fell to 23%. In the control group, the cor-responding rate was 62%.

The Efeot of 4L? and VU-2 PrearatIons on the Survi••l&%Ie of gals irladtazed WlI~h a 700 & Dole From am O0--•u

t I I I&.:U ,ii

A = Variant; B = Total number of animals; 0 a Number ofperishing animals; D = Absolute number.

Prom the data of Table i we can conclude that thePLP preparation has a protective effect against Ionizingradiation. The effect of the PLP Is intensified when it iscombined with the VB-2 preparation, which is oonfirAd by

2

statistical treatmentl in the first came p 4 I i0"3, and

in the second, pe 2,10-3.The data showed that the condition of theblood

of the experimental animals was much bettýer than that ofthe control group. The content of hemoglobin in the experi-mental rate 5 days after irradiation was 103-1051 of theinitial level, followed by an insignificant drop to 95-96%.By the 30th day following irradiation, the hemoglobin con-tent had risen to 100-104 of the initial level.

F'ipure 1.* Variations in hemogram of rats irradiated fromSCo• 0 source (700 r dose) with combined protection (PLP +VB-2). Averapge data, a -- experiment, b -- control. 1 --ho•on,2-ertoyee -- etuooesthrombocytes. A = '• of initial level: B = Days after irradia-tion °

Ir. the control rats, starting on the 5th day follow-in• irradiation, there was a grdual decline in the hemo-4lobir content in comparison to the initial level; it reachedits lowest values (471) on the 15th day after irradiation.By the 30th day the hemoglobin content for the control•'roup wae 5E•. The number of erythrooytes in the bleod ofthe control rats from the 5th day after irradiation drppedgradually, reaobiin• s minim~um (60% of initial level) on the20th dey after irradiation. In the group 1 rats, in alltime Intervals (starting from the 5th day) following irra-diatiorn, the increase in the number of erythrccytes incomparison with the control mp~cimens tame statisticallyevident (with the exception of the 30th day followinpirradiation).

1110

With combined protection, the survival rate wabhigher; on the 10th, 15th, and 20th day after irradiation,regeneration was more intense than with the administrationof PLP alone. The increase in the erythrocyte count, startingon the 5th day, is statistically evident in all time inter-vals following irradiation (see Figs I and 2 and Table. 2).

It must be noted that the decrease -in the absolutereticulocyte count in the experimental group, cale later thanin the control group. 5 days following irradiation, boththe control and experimental specimens exhibited a sharpdrop in the absolute reticulooyte count; for the experimentalgroup, the count was. 17-251'of the initial count; the cor-responding figure for the control group was 8.374. The re-generation process in the.experimental grouy began soonerand was much more intensive than in the control group.4ith combined protection, the regeneration on the 10th,15th, and 20th days following irradiation was more intensethan with PLP protection alone.

40

40

Fii.mre 2. Variations in hemogram of rats irradiated froma Co0 source (700 r dose) with ?i? protection. Averagedata, Notation same as in Fig 1 A = • of initial level;B = Days after Irradiation.

Fluctuations in the thrombocyte count over theentire observation period did not exceed normal limits.In the control group, the thrombocyte count from the 5thday following irradiation began to fall. reaching thelowest levels on the 15th, 20th, and 3oth day followingirradiation (see Table 2). The increase in the thrombocytecount in the experimental animals in all time intervalsstarting with the 5th day after irradiation was statistic-ally evident in comparison with data for the control group.

With recard to the leucocyte count it must be notedthat its in~rease became evident statistically just one dayfollowing irradiation.

The PLP preparation extracted from tea leaves, incontrast to BLP, does not produce a stable and clear varna-

4

-. :. _...V, , + . .'I+t +.+-_+ai _'- 1.0A

4M'

---- -

VA -l -A 21 4 00

W, 4-t10 ....:. - i

alt0 46 -0 a*

INI

d? $4P-4

+1 + "°5

tion in experiment irs. comparison with control in the pictureof the leucooyte *eriLes.

Thus, we may conclude that the lipopolysaccharidepreparation extraotedk from tea leaves has a protective actionagainst radiation, irmcreasing the survival rate of Irradia-ted animals anid prodimcing an increase in the hemoglobin coný-tent and the erythroc~yte retioulocyte., and thrombooytecounts. The effect cff this lipopolysacoharide preparationupon its combined uses with VB-2 (the vinylbutyl ether polymer),As distino.t from the bacterial lipopolypacharides, the tea-leaf PLP has the abi3.ity to exercise a protective effsect onthe hemopoietic funcoitin and to stimulate erythroa and thrombo-cytosis without subeltantially and stably altering the pictureof the leucocyte ser!L-esBiological Physics rinatitifte Receivedof the Academy of Sc!.erices USSR 12 April 1962Organic Chemistry Iumatitute imeniNfl. Zelinukiy of thin Academy ofSciences USSR

Reof ereogg.

1. 11 , romme 0, Lumederitz, e~t al,, Pharm-c. actj helv.,, 33,391 (19585.

2. 0. Westphal, Al nt Faor 98, 789 (1960).3. Z-.V. Yermol. eva G.Te, Vayeeg et al., ________o

F-o 6,ý 46 (199811, .. ybra lAtboi4. E. Proicke, V. Tr-~obst I G. Schumacher, Schweiz$ meds

Wocyhono. 88, -111 28, 91 (1958).5. TYeer- ohn k. . Vogel, Arch. I-, X11n, u. exp. Dermatol.,

210, 31 (19605.6. Ch. Borrel J. ?-Tel. A. Vanotti, Schwe~z. med W'ochenschr.,

91 , No 1, (196 1).7. A.A. Tunis W.J. Harrington, J.- Lab slid Olin.-Vied., 55,

No 4, 584 ?1960)8. J.G. Tloward, J., 71tho1. and lagteriolg,, 78, No 2, 465

(1959).9. AI. 'Braude, G,=e, Vayeberg, DO (Dokiady Akademii Yauk

-- Reports of tIx~e Academy of Sciences USSR), 136, Yo 2

10. B.Y. Gladyshev, t t tai~ lceilr-CnrlL

11...,N Gadysihev, 145, No 4 (142).12 .. Sna Y, troei in the colloctiýý V111minnvvelegrl v4'eir Asouce ,No 4 1959, 1

13. fR.As r~ard, 4kC2 atl~ *MI6 5, 117 (1014. R.A. Canard, J. Amj Phs171 165, 375 (1951)10,106030: 2456-8

6

ON TH SMZCTIVE SOCT OF MTRASONZC WAVES ONTHE MOLECULAR STRUCTUIM OF INSULIN

[Feiloewif Is the trauulatiou of a nrtiole byIs Ye. 3lpifer mnd L. T. Stekollldkov, in theRusian-lnuguage periodioal koklaay Altdent Nal(Reports of the Aaadmy of Soienoee US Q, No 4,MoSoCw, 1962, pp 929-932.]

(Presented bw kcaderAcian A.T. Onarin 2 July Iq62)

r. -.n ultrasonic f•Id, nroteins and enzrymes splittr into lr. Ivleual fragments of considerable 'nbleculalrw ~ht ( 1~ .. vd 2). .'his rrocess occurs only when the',rot•.in. co!';t1on thus treated 's saturated With oryv-er. (alr)or arg'o- I,-" .f the ¶olecular weimht of the rroteir is overPO,O0, . 'o ntart with, we had only indirect indications thaturon the "!lrasoric treatment of a proteir solution in the•r~nc cf ovivrter, It is r'oesible to havc not only the split-+in• of tke protein molecules into relatively larpe molecularf"•a; ents, *but also the removal from the proteir. moleculeof ir.diviiaul Pmindscids or'.minor -eptides (refs 3, 4).Iere in the case of insulin it was established that thehormonal l'xinctio,n of this pro~eir is intensified depending.on the nnture of the pas present in the treated so'!ution(r'Cf ý). The most obvious data 'on structural changes werecbtnined by us through the further study of uhf sound onwater solutions of insulin.

""he choic 'of insulin was dictated by the fact thatthe orrr clf distribution of amfnoacid residues in its mole-cule bha beer. firmly established. The molecular weight ofinsulin is ý.000 or 6000 X 2, which ercludes the possibilityof Molecule stlittin.• under the action of ultrasonic Wavesinlo relatively large frapments (ref 5). In addition, it isfhirly Ftsy to determine the biological activity of thehormcne stulied.

At is known the insulin molecule consists of twopolypeptide chains (A •nd R) connected by disulfide bridges(see di-rpn) .

MHR NHS NH•

Fa.Cr,.fel3ure.aaJetTu.t a.a" - -U •.At.a.*U.UTU -4P@fU-A

DiaRA gf BIgoaold Reosdue Distribution in Insulin Molecul6sLose English version p 91.

In the A-chain,i the N-terminal bond aminoacid isglyoine, and the 0-terminal bond -- amparaginel in the B-chain, these are phenylalanine and alanine, respectively(refs 6, 7), To determine the N-terminal bond groups, weemployed a method suggested by Sanger (refs 8, 9) based onthe fact that dinitrofluorobenzol (DNMY) in a weakly-basicmedium interacts with the free NH2-groups of the protein toform dinitrophenyl derivatives:

P

NO, + NHOCONH.'.-'

NH.CH.CO-NH ...

NO,The resultant product was subjeoted to acid hydroly-

sis in sealed ampules at 1000 in 12 N HC1 for 8 hours. Al-most all of the dinitrophenol aminoaoid derivatives (DNP-aminoacids) were extracted from the hydrolysate by means ofother. The free aminoaoids were removed from the ether ex-tract by solution In water, while the residual other con-taining DN?-aminoaoids was dissolved in alcohol and appliedto ohromatographicpaper. The solvent employed was butanolsaturated with 0,1 ammonia. The indicators were alcoholsolutions of DNR-aminoacides

The insulin we used, treated by the described method,produces two spots on the ohromatoFram (Fig 1) whose positioncoincides with the spots of the DNP-glyoine and DNT-phenyl-alanine employed. These results confirm the data found in theliterature which indicate that the N-terminal bond aminoacideare glycine and phenylalanine (refs 8-10) (the resultingdinitrophenol bpat, a side reaction product, was brought outby placing the chromatogram into concentrated HOl vapor for5-10 minutes).

Other results were obtained in the study of sound-treated insulin preparations. The treatment was applied to

S~8

04 P

F4

m I

e-H

04

C~Cc)

to r-4HtN ~ I d 04

0

E-H 40

A r4 0

I A I

H PI

0 0d

9fI

an O,3% water solution of insulin for 3 houri with an ultra-sonic wave intensity of about 1O-12 watts/omdd (frequency --740 kilooycleu/seo). The irradiated solution was saturatedwith oxygen, argon, or hydrogen,

1.14

-tJi

--------~~ - --- ~k 0 1 _3 K

pip, 1 Fig 2'Figure 1,* Chromatograms of N-terminal bord DINP-derivativearninoacids of Insulin molecules. a -- DN'-phenylalanine;C' -- DYNP-rlycine; 6 -- insulin not subjected to ubf souri3treatment ( lower spot -- DNP-glycine, upper epot -- DNP-phenylalanine); 1L -- insulin solution sound treated in pre-sence of 0 (lower spot -- DYP-glycine, upper spot -- DPhistidine)Hb -- DN?-glycine; v, -- flr'P-histidine,

Figure 2. Chromatograms of N-terminal bond DNP-derivativeinsulin aminoacids for insulin subjpoted to uhf sound treat-menit in presence of argon. a -- DYP-phenylalanine + flNP-glycine; I' -- DX?-alaziine; u - DNP-cystine; 1. -- Insulinsolution treated in presence of Ar (lower spot -- DNP-aystineor fNII-cysteine acid, upper spot -- DX?-phenylalanine).

P'igure I is a chromatogram of an alcohol solution of flNP-derivative arninoacids obtained after appropriate hydrolysisof the Insulin sound-treated in the presence of oxygen. Thechromatograms invariably revealed a DNIP-glycine and a flNT~-

10

histidine spot. Consequently, under the action of uhf wavesin the presence of oxygen, there is a cleavage of the peptidebond between the glutamine and histidine in the B chain.Further, the tetrapeptide P-al-Val-Asp(XH2)-Glu(NH 2 ) breaksaway from the insulin molecule. It should be noted that thechromatogram did not reveal a spot for DNP-phe.nylalaninewhich should have been formed as a result of DNB interactionwith phenylalanine which is the N-terminal bond aminoacid ofthe removed tetrapeptide. Apparently, the phenylalanineincluded in the tetrapeptide undergoes chemical transformationsas a result of uhf sound irradiation which deprive it of theability to react with DNPB.

It is Interesting to note that in the, case of insulinsolution sound treatment in the presence of argon, it is the:pentapeptide from the A chain of the hormone molecule ratherthan the.'tetrapeptide from the B chain which splits off.This is indic&ted by the chromatogram of Pig 2. Here the in-

I I I I I

Figure 3. Chronatograms of aminoacids obtained as a result ofthe action of carboxypeptidase on insulin before and afterits sound treatment.,a -- untreated insulin; d-- insulintreated in presence of oxygen (upper spot -- alanine, lowerspot -- aspartic acid); S -- insulin treated in presenceof argon; v -- insulin treated in presence of H2 ;7 -- alanine;e -- aspartic acid.

sulin treated in the presence of argon and further processedwith DNPB, produces two spots: DNP-phenylalanine and DNP-oystine. In other words, in the presence of argon under theaction of uhf sound, there is a rupture of the polypeptideA chain with the appearance of cystine or its derivatives(DTP-cystine and its derivatives are close in their Rf) asthe final N-aminoacid. In a number of oases, the chroma.togramalso revealed a glutamic acid stot. We used the dinitrofluoro-benzol method unsuccessfully in an attempt to detect changesin the structure of insulin upon its sound treatment in thepresence of hydrogen. Changes were detected, however, withanother technique permitting the determination of the C-terminal bond aminoacids in the insulin. For this purpose

11

we used the oarboxypeptidise method* Oarborypeptidase splitsup largely those peptide bonds in the protein which are adjacentto the d-carboxyl group.

The insulin solution (50 mg of protein) (pH 7.8) inthe presence of the oarboxypeptidase enzyme was incubated at37 and 251: for 4-6 hours. Triohloraoetio acid (20) wasadded following inactivation of the enzyme and the solutioncentrifuged. The fluid over the preoipitate was decantedand evaporated in a vacuum (at 40 ) until dry. The dryresidue was dissolved in water and ohromatographed (solvent-- butanol (CH 3 COOR- 2 0); developer -- ninhydrine),

The untreated insulin produced a single spot on thechromatogram (Pig ý)j this belonged to analine, as indicatedin the literature (ref 11). Prom the same chromatogram we seethat the preparations of insulin sound-treated in the presenceof H2, 02, or Ar along with the alanine spot likewise yieldan aspartic acid spot. This indicates that in the ultrasunicwave field at the end of the A-chain there takes place thedeamidation of the aspartic aoid which is split off from theprotein by the carboxypeptidase. The deamidation of the as-paragine (probably glutamine as well) was accompanied by therelease of ammonia, which was detected by the Nessler reagent.

Figure 4. Ohromatograms of asparagine before and after

sound treatment in the presence of 02 (asparagine concentra-

tion -- 10 mg/ml; length of treatment -- 10 hours; ultrasoundfrequency 10 watts/cm ). a -- aspartio aoidl t- - sound-treated asparagine (upper spot -- aspartio acid, lower spot-- asparagine); * -- asparagine.

It should be noted that asparagine under the actionof uhf sound can actually undergo deamination, as indicatedby the chromatogram (rig 4).

12

It can also be surmised that the glutamine at theend of the polypeptide obtained in the sound treatment ofthe insulin solution underpoes deamination in the uhf wavefield with the formation of glutamic acid (Pig 2).

Thus, uhf sound wavea, dependinn on the nrtuve ofthe gas present, produce ruptures I-n the polypeptide chainat strictly flved T)oin+s of the insulin molecule: in the•rosence of oxygen, a tetrareptide splits off prom'the K-endof the '-chain; in the nresence of argon, a pentapeptideSplits off from the "-end of the A-chain. !n the presence -ofhydrogxen, we note only the 6eamination of asparaAine on theC-end of the k-chain of the insulin molecule* the deaminationof the qsparapine is likewise observed with the sound treat-tent of insulin in the presence of 02 and Ar. The latter

fact indicates that asrarnine deamination at thr C-end of theinsulin occurs as a result of both its interaction with 02and 1. (activated in the ultrasound wave field), and as a

result of its activation by OTT-radicals whose formation iscatalyzed in the presence of ar-on (ref 12).

The srlittinr off of the nentareptide from the A-chain in the presence of armon with the participation ofthe OCl-radiaclsoccurs with the possible selective reactionof the lattpr with the aminoacid qlutamine and cystine resi-dues. The oy".en actiwvted i', the ultrasonic wave fieldapparently transforms chemically the Pmvnonacid rlutamine an,11'istidine r-esidues, as a result of which a tetrapeptide splitsoff from the P-chain of the Inculin molecule.

9iolo-ical Physics Tnstitute of t1i- ReceivedAcndemy of Sciences 1TS5R 30 June 1062

leefrenoes

1. *.Ye. =]'piner, G.A. . eborin, O.Y. Zorina, oiokhim•al-(?ooehemistr0), 24, P117 (1959).

2. I.Ye. rl'piner, Akunticheskti Zhurnal (Journal of Acous-tics), 6, 399 (19•60).3. I.Ye. Bl piner, BTulleten' eksnerimenta!,noy biplorri i

meditsynv (Journal of 7xperlmental Rloloqy and 7Yedicine),9, 207, 1950.

4. T.Ye. Ml;pIner, 0.Y. Zorina, "Riofizik. (niophysics), (,573 (1960).

5. ". Schmid, Ze. phys. Chem , I M6, 113 (1 940).6. F. San~er, 0. Thompson, 'Iocham J ,53, 353 (1953).7. P. Sanger, "..rTunpy, biS, 49, 49' (1951).

F 1. SanjRer, ib d., 39, 507 (1945).9. A. %insburrý, F. Schachman J Bild Chem , 235, 10'ý (1960).

10. F. Sanrder, ?iochem.J,, 4 f26 (1J4.,,11. J,. Len . U'iochim. biophys. acta, 3, 367 (1949).12. T.Ye. TL'riner, A.V. Sokol'skaya, DA.., 129, 202 (lq 5 9).

Io •,106CS0: 24536-9 13