AD- 787 398

.'1HE EFFECT OF FORMALDEHYDE ON THE- -ACTIVITY OF ANTHRAX

G. A. Maksimova, et al

Foreign Technology DivisionWright-Patterson Air Force Base, Ohio

18 September 1974

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THE EFFECT OF FOITATr')EHYDE ON THE ACTIVITY OF ANT11RAX

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G. A. Maksimova and V. F. Runova

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EDITED MACHINE TRANSLATION

.TD-MT-24-l357-74 l8 September 1974

THE EFFECT OF FORMALDEHYDE ON THE ACTIVITYOF ANTHRAX

By: G. A. Maksimova and V. F. Runova

English pages: 7

Source: Zhurnal Kikrobiologii, Epidemiologii iIimunobioloqii, Vol. 47, Nr. 10,1970, pp. 59-62

Country of Origin: USSRRequester: FTD/PDTRThis document is a SYSTRAN macaine aidedtranslation, post-edited for technical accuracyby: Charles T. Ostertag, Jr.Approved for public release;distribution unlimited.

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. OCT 3 1974

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THIS TRANSLATION IS A RENDITION OF THE ORIGI-NAL FOREIGN TEXT WITHOUT ANY ANALYTICAL OREDITORIAL COMMENT. STATEMENTS OR THEORIES PREPARED BY:ADVOCATED OR IMPLIED ARE THOSE OF THE SOURCEAND DO NOT NECESSARILY REFLECT THE POSITION TRANSLATION DIVISIONOR OPINION OF THE FOREIGN TECHNOLOGY DI- FOREIGN TECHNOLOGY DIVIS'ONVISION. WP.AFB. OHIO.

FTP.MIT 24-1357-74 -Dtele set 19 742.

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FTD-MT-24-1357-74 ii

THE EFFECT OF FORMALDEHYDE ON THES•. ACTIVITY OF ANTHRAX

G. A. Maksimova and V. F. Runova

State Control Institute of MedicalBiological Preparations imeniTarasevicha

(Submitted 27/I, 1970)

The anthrax allergen, isolated by the previously-described

procedure (Runova and Ulanov, 1966), Is a protein-polysaccharide-

nucleic complex which contains up to 80-86% of protein. The

carrier of the specific activity is the protein component of the

preparation (Runova and Rudnev, 1968). For the purpose of explana-

tion of the chemical determinant groups of anthrax allergen it

was iodinated under varied conditions, which led to the significant

loss of activity along with full blocking of phenol groups

(Maksimova and Runova, 1969). In connection with this the assump-

tion was made about the significant role of tyrosine in the blo-

logical activity of anthrax allergen. Earlier lRunova and Rudneva

(1968) noted a lowering in the specific activity of preparation

following formalin treatment. Some researchers (Zaretskaya, 1959;

Mus~ykc, 1957; Gavrilenkova and Runova, 1967) connected the

lowering in the activity of allergen following formalin treatment

only with the blocking of amino groups, assuming that the reagent

acts selectively on them. However, it is necessary to keep In

FTD-MT-24-1357-74 1

mind that th action of formaldeh de on the protein molecule depends

on the conditions of the reaction - the duration of the interaction,

temperature and pH of the medium (Putmam, 1956). The short-time

action of reagent on protein at room temperature and pH 7.0-8.0 is

more specific in relation to the amino group. In this case there

is compensation of formaldehyde with the amino or imino group and

the formation of methylamines. However, during the isolation of

the modified product the reaction flows in the opposite direction.

The prolonged treatment of protein at 25 or 370 and pH 3.0-9.0

leads to the irreversible or partially reversible reaction with

the formation of cross methylene bridges between the amino group,

on the one hand, and other groups, in particular phenol and indole,

on the other hand.

Thus formaldehyde is not a highly selective reagent on the

amino group and therefore with a lowering in the activity of

allergen it is necessary to consider its secondary interaction with

other groups of the protein molecule. In the present work an

attempt is undertaken to reveal the functional groups of protein

which are responsible for lowering in the biological activity

of anthrax allergen following formalin treatment.

The anthrax allergen was exposed to formaldehyde according

to the procedure of Takeya and Mifuchi (1954). The allergen were

dissolved in borate buffer at pH 9.1 with the addition of formalin

of up to 1.5 and 20% concentration of formaldehyde in the solution.

The control sample did not contain formalin. The samples were

maintained at 370 for 5 days, dialyzed for 2 days against top water,

and for twenty-four hours against distilled water, and then

lyophile dried. Taking into consideration that the reaction of

formaldehyde with proteins can be reversible (Takeya and Mifuchl,

19541), half of the preparations obtained was dissolved In distilled

water, heated at 1000 for 30 min, then dialyzed and dried, as is

described above. In all samples the relative content of free amino

FTD-MT-24-.357-74

_groups was determined by the ninhydrde method (Kebot and Meyer,1968), the content of tyrosine - according to the method of Polina

(Kebot and Meyer, 1968), and the content of tryptophane - accordingto method of Spies and Chambers (1949).

hUV spectra of the absorption of modified preparations andattheir corresponding control allergens: 1 - controlallergen; 2 - control allergen, heated at 1000 for 30 min.;3 - allergen, processed by 5% solution of formaldehyde;•4 - allergen, processed by 5% solution of formaldehyden and heated at 1000 for 30 min.

In three preparations, treated with the

indicated solutions of formaldehyde, theSI content of the above-enumerated groups was

reduced. In this case the quantity of tyro-.. ... sine and tryptophane wa- decreased in them

to 30 and 70% respectively and did not de-

pend on the concentration of formaldehyde. The number of amino

groups in the preparations treated with formalin was decreased

with an increase in the concentraition of formaldehyde, comprisingcorrespondingly 43, 37 and 36; of the content of amino groups in

the control allergen. After the heating of the formalin-treated

preparations the content of tyrosine in them was virtually com-

pletely restored (97-100%), the content of tryptophane partially

restored, and it comprised 50% in all preparations. The concen-

tration of amino groups also increased somewhat, comprising 65,

53 and 148% respectively for preparations treated with solutions

of formaldehyde.

The spectra of absorption in ultraviolet were determined

for each preparation with the aid of an SPFi* spectrophotometer.The allergen was dissolved in borate buffer (pH 7.4), the con-

centration of protein in solution was in this case equal to

100 lAg/m1. The figure gives the spectra of absorption of the

FTD-MT-24-1357-74 3

preparation, treated with a 5% solution of formaldehyde, and the

.aame preparatioan after heating• ... and also their control allergens.

The maximum of absorption in all cases was observed at 265-280 nm.

The magnitude of absorption in the preparation heated after treat-

ment with a 5% solution of formaldehyde was higher than in thepreparation, not subjected to heat treatment. Analogous resultsare obtained for the preparations treated with I and 20% zolutionsof formaldehyde. It may be concluded that the formalin treatmentled to dimunition in the content of cyclic amino acids in the

allergen, and the heating of the formalin-treated preparation ledto the partial restoration of the total content of tyrosine andtryptophane. Thus spectrophotometric data qualitatively confirmedthe results of the chemical analysis of the modification ofpreparations. For the purpose of the determination of the specific

activity of allergen it was dissolved in sterile borate buffer

(pH 7.4) up to a cc.Icentration of protein of 500 ug/mZ (concentra-tion of protein was determined spectrophotometrically by the method

of Wadell, 1956). Solutions in a volume of 0.1 ml were introduced

into the end of the lateral surface of the body of sensitizedguinea pigs weighing 350-400 g 21 days after their immunization

with live STI-I vaccine In doses of 25 million spores. The

activity of the modified preparations was evaluated in 24 hours

according to the extent of the area of hyperemia and compared

with the activity of the control sample. All the preparations

were specific, since in nonimmunized guinea pigs a skin reaction

was not caused.

The specific activity of the formalin-treated allergens intwo parallel experiments turned out to be lowered to an identical

measure - it comprised approximately 50% of the activity of the

control allergen (Table 1). Lowering in the activity of each

preparation in comparison with the control is statistically

reliable, since with T>3 its probability comprised 99%. Since

the activity of all formalin-treated allergens turned out to be

equally lowered with a different content of amino groups in them,

•4

* -R

Table 1. Change In it can be assumed that the lowering inthe activity of biological activity, apparently, is notanthrax allergenas a result of connected with blocking of the latter. Sftormalin treatment_____ treament At the same time a correlation was

8 t 3I ofet observed between the change in activity

and the content of determined cyclic

r •44= amino acids - the activity and con-'" centration of tyrosine and tryptophane

,,3.01 In the formalin-treated preparations

IM 3s12j11.7 4.91ISI was maximally reduced already with the

- 30 I0.31 4.0 1 W lowest concentration of formaldehyde.

S] IX n. 1 S7 1 SO5I M 42.51u1.01 3.6 53., . Not in one of the preparations,

20 S Sj 37, iso ... heated after treatment with formaldehyde,IN 4S. 1 19.0 1 3.9 C was a statistically proved lowering

* ote. In the upper row- tree Sl the ,rrst.ihe

loer ! ge resurq3 of # of allergen activity observed (Table 2).SorH ex~pnt (in tacr.el eeraeA.TTere vere r The restoration allergen activity in. gu nea pigs/,

the heated formalin-treated preparations

"apparently cannot be connected with anincrease in the content of free amino groups, since the concen-

tration of the latter rar•led from 48 to 65%, In this case theactivity of all prepirations was restored virtually completely.Besides this, the ccntent of amino groups in the allergentreated with a 20% solution of formaldehyde and warmed thoroughly,

and In the allergen treated with 1% solution of formaldehyde

(not warmed thoroughly), were comparable (48 and 43% respectively),

nevertheless the activity in the first case is equal to the

control, but in the second is lowered by 50%. The results obtainedmake It possible to conclude that the amino groups of anthrax

allergen apparently are not essential for its activity. This

* proposition is confirmed by the results of selective N-acetyla-

tion of the anthrax allergen: with acetylation of the latter

by acetic anhydride in a half-saturated solution of sodium

acetate 801 blonklng of aminc acids occurs, nevertheless thebiological activity of preparation is not changed (Tw0.8).

5FTD-MT-24-1357-7 5

Table 2. The statistteal Interpretation of the activity of t•hepreparations, heated thoroughly after formalin treatment.

7IConcentra- Characteristics of preparationstion offormaldehyde(in 5) m a m T

256 34.3 13

261 65.7 27

_222 44.7 __17_1I J_1 I i

218 51.8 20 1.6

197 63.2 26 1.7

213 66.8 25 1.520 '

Z02 67. 27 1.5

The fact that the full restoration of phenol groups is

accompanied by the full restoration of activity after the heating

of preparations, indicates the dependence of the specific

activity of anthrax allergen on the content of ty-osine. Since

the activity of the heated preparations is retained in the

presence 50% indole groups, apparently the latter are not

essential f~r the biological activity of the allergen.

In conclusion it is necessary to note that the results of

this work, which indicate the connection of the specific activity

of anthrax allergen with the content of tyrosine, confirm the

assumption made previously (Maksimova and Runova, 1969) about

the possible participation of tyrosine in the mechanism of the

allergic reaction.

Conclusions.

1. The lowering of the specific activity of anthrax

allergen following formalin treatment is connected with the

action of formaldehyde on the phenol groups.

FTD-MT-24-1357-74 6

2. Amino and indole g-raupa are not essnti-al" for *-h4 blcos

logical activity of the preparation.

BIBLIOGRAPHY

raaF wP Aeu oawOXu .n t a a 10..P xsaa0.3.modm .le'7.s W c. c21.-Mamemo o. A_ A anc * aaaxopya I- n6~eahmit ara onHAmofl staj. O -rs. 19.- Pyanma & t P 13 z.ae. a. P-A. XMUpo-

a,.1 7 . c. 139. -Own ateTaw.- .. M-I0.e- P.N-saaa S. *_. Y. A&m uasa A. A. S a.1. Mnpus esmrMltvtttsao U0160~11i ramas" .1. A. ?apnaý.M4.lac6fC.-Ka o E M eike p 314 8 a-.- 3omeveruumns a unawjlam..

M.. 19.e 0.--Spies J. R.. ChamberT D. C. Anatyt. Chem.. to. w. 21.* ;. 124. -1 nVT a IA. Biln.: &easno i'm pea. r. Hivaer. K_ b>*x-i AL. INA. 4c.31&.-

a khe ya 9.. MIr.chi I. En•y-otboa, |1,,25.v6. V. 366. -c-Waetle I .- J.,I.l.sL. tin. fWd.. BM6, v. a p. 311.

The authors studied the effe0t of 1.3 &4d 20P. Iorul4Jehyde Soluliont Mj H 9 1 oanthebiakieal vliviivt of ftl anthrax alklrn. The actiity o0 1a'1 1bt unal-d pretlmattiodecreased n lJk b"k aprnxsmately W,1 and in-sihdq oede f fil e cUnceel'almoa otftatliv&•. Ritdution of tvro-'4q and tryvptphane cntrent •r•-c•uird in analiocow wav(by 30 ad :W.. rr.-ptivr% t-is .a the Saln thnt lie amontl of tree aminou gupe dimrtilio-eWiit &italion of lont-larhsdet-otiialintt o loniniod prepratioe at i3WCifu 30 ninute Wle to copkde re-toration of specific arlititv in all the preenratiom, ulvo:with Thiltee resortzn of the content .1 phno C hiroe3- The 'om-ntration of amino, goupsand t-yptophaie in thon pleparationi, atd 0 to 60% of their coocttratton in the crudeallerglen.

The emiss obtlained led to a conclusion lthat the aivifv of antrealh akhes was ua-elated with thlt eontert %t pof nlw Lgoup a-wma! ninA ami t!an l t oops wrie appartntlyoi nlnlwanii cie Ionr bsnkcical allisaily of thepeqeprmatiran

FTD-MT-24-1357-7q 7