COMPLEX FORMATION BETWEEN BASIC ANTIBIOTICS AND DEOXYRIBONUCLEIC ACID IN HUMAN PULMONARY SECRETIONS

(1)

COMPLEX

FORMATION

BETWEEN

BASIC

ANTIBIOTICS

AND

DEOXYRIBONUCLEIC

ACID

IN

HUMAN

PULMONARY

SECRETIONS

Joseph 1. Potter, M.D., Ph.D., LeRoy W. Matthews, M.D.,

Samuel Spector, M.D., and Joy Lemm, BA.

Department of Pediatrics, Babies and Children.s Hospital, Western Reserve

Unwer.sity School of Medicine, Cleveland, Ohio

(Submitted March 19; accepted for publication May 12, 1965.)

Presented in part at the Thirty-second Annual Meeting of the Society for Pediatric Research, Atlantic

City, New Jersey, May 8-10, 1962.

This investigation was supported by grants from the U.S. Public Health Service (AM-08305 and

AM-0385), and a grant from the National Cystic Fibrosis Research Foundation.

ADDRESS: (J.L.P.) Bal)ies and Childrens hospital, 2103 Adelbert Road, Cleveland, Ohio 44106.

714

PEDIATRICS, Vol. 36, No. 5, November 1965

T

HE Precil)itatioil of Ilucleic aci(ls by

streptomycin, a basic antibiotic, was

reported by Cohen in 1947.’ More recently,

the same author publisileci a method for the

isolation of deoxyribonucleic acid

(

DNA)

and of nibonticleic acid

(

RNA

)

from

bac-terial extracts with the use of the same

anti-biotic.2 In our laboratory, human pulmonary

secretions were initially collected either by

direct aspiration from the tracheostomies

of laryngectomized patients, or by the

transfer of expectorated material from pa-tients with cystic fibrosis and

bronchiec-tasis illto prepared vials containing a

mix-ture of neomycin and polymixin. 4 Tllese

antibiotics were introduced to minimize bacterial alteration of tile pulmonary glyco-proteins. It was Iloted that after

lyophiliza-tion of the secretions and reconstitution as

a 1% suspension, the supernatant solutions

obtained by centnifugation demonstrated

antibacterial activity in samples from the

broncinectasis and laryngectomy groups.

However, comparable supernatants from

the secretions of patients with cystic fibrosis

showed no antibacterial activity. It has been subsequently demonstrated that the absence of antibacterial activity in

the cystic fibrosis secretions is directly

re-lated to the larger amounts of DNA present

as compared with tile other two groups of

secretions.14 Tile present work is concerned with a sttidy of complex formation between

basic antibiotics and DNA in human

pul-monary secretions and in simple systems

containing only DNA and antibiotic.

Bind-ing afl(l PreciPitatioll of RNA iy tllese

antibiotics is also described. The influence

of pH, ionic strength, and nuclease on tile

complex have been studied. The minimtim

chain lengtil of polynucleotide required for precipitation has been determined. The

possible significance of the reaction in

whole pulmonary secretions and in relation

to tile treatment of purtilent foci of

infec-tioll is considered.

M ETHODS

DNA was Prel)arel from various sotirces

by the method of Kay, Simmons, and

Dotince.5 RNA was prepared from yeast

by the metilod of Kay and Dounce.C

Trans-fer RNA0 and deoxynibonticlease I

(

DNase I

) f

were commercial products. A series of polynticleotide chains composed exclusively of pynimidine nucleotides was prepared

from calf thymus DNA by the method of

Burton and 2 A sample of DNA

was treated with Burton’s reagent for 16

ilours at room temperature. Formic acid

and diphenylamine were removed by ether

extraction, and the pH of the aqueous layer

adjusted to 8.5 with ammonium

hydrox-ide. The mixture was chromatograpiled

* Nutritional Biochemical Company, Cleveland, Ohio.

(2)

4.

l2

>. 1.0 H

En

z 0.8

0

-J

0.6

0

H 0.

0 0.4

02

\

\\

\

220 240 260 280 300

ARTICLES

on a Dowex-1X2 coltimn, 15 by 0.9 cm,

in the formate form. Elution was effected

with increasing molarity of ammonium

formate at pH 4.5. Chain lengths were

determined with snake venom

phosphodi-esterase as previously described.5’

Human pulmonary secretions were

col-lected in vials containing 40 mg neomycin

sulfate and 400 .g polymixin stilfate, or in the absence of the antibiotic mixture. There were approximately 60 gm of whole

pulmonary secretions in each vial. The

secretions were lyophilized, and the dried material suspended in water at a

concen-tration of 10 mg/mi. After stirring for

one hour at 5#{176},the suspension was

centni-fuged at 13,000 RPM for one hour, and

the supernatant removed. The sediment

was washed with a little water and

re-centrifuged as above. The combined

supernatants were designated water-solu-ble fraction

(

WS ), and the sediment

designated water-insoluble fraction (WI).

The antibiotic activity of the

(

WS) fractions was assayed by wetting

Wilat-man No. 3 MM filter paper discs in the

solution. Tile discs were lightly blotted to remove excess fluid, and then applied to agar plates which had been previotisly streaked with strains of staphylococci sen-sitive to the particular antibiotic. The plates

were incubated at 37#{176}overnight, and a

clear area of at least 2.0 mm around the

disc was interpreted as evidence of anti-bacterial activity.

RESULTS

The

(

WS

)

fraction of ten specimens of secretions collected in vials witil tile

anti-biotic mixture from the laryngectomized

group and of six bronchiectatic specimens exhibited antibacterial activity. Only two of

thirteen samples of the

(

WS

)

fractions of

the cystic fibrosis secretions similarly col-lected exhibited antibacterial activity. The

(WS) fractions of a series of samples from the bronchiectasis and laryngectomized groups collected in the absence of the anti-biotic mixttire did not exhibit antibacterial activity. This indicated that tile secretions

WAVELENGTH mji

FIG. 1. The ultraviolet spectra of the (WS) fraction

and (WI) fraction from the pulmonary secretions

of patients with cystic fibrosis. Open circles =

(‘tVS) fraction; closed circles = ( WI) fraction after solubilization by treatment with DNase I.

had 110 inherent antibacterial activity, at

least under these experimental conditions.

The failure to demonstrate the activity of

added neomycin-polymixin in the case of

the cystic fibrosis secretions indicated

eitiler inactivation or removal from the

soltible fraction.

On the basis of Cohen’s early study

con-cerning tile precipitation of DNA by strep-tomycin, it seemed likely tilat tile larger

amounts of DNA present in the cystic

fibrosis secretions1 might account for the

observed phenomenon . The ultraviolet

spectrum of the (WS

)

fraction of samples

from the grotip of patients with cystic

fibrosis is shown in Figure 1, and exhibits

a peak at 275 ., characteristic of protein.

The

(

WI

)

fractions from these secretions were stispended ill 10 ml of distilled water

and 2.0 .g crystalline DNase I added to

each sample. After incubation for two hours

at 370, the suspensions were centrifuged at

(3)

I-z

I-z

LiJ

a-(I)

z

a !00

80

60

40

20

0 20 40 60 80

Antibiotic

Neomycihl*

Polymixin

Kanamycin

Colistint Streptomycin

Jg NEOMYCIN BASE

FIG. 2. The precipitation of calf thvmus DNA by

neomycin.

spectrum of the resultant superriatant

solu-tions is also shown in Figure 1, and exhibits

a peak at 260 p., characteristic of nucleic acid. Assay of these supernatant solutions

showed distinct antibacterial activity in all

of the specimens examined. Next the (WI)

fractions of six samples of cystic fibrosis

secretions collected in the

neomycin-poly-mixin mixture were suspended in M sodium

chloride and stirred for one hour at 25#{176}.

The suspensions were centrifuged as above,

and the ultraviolet spectrum of the

resul-tant supernatant solutions was found to

TABLE I

ANTIBIoTIcs PRECIPITATED WITh DNA

Molar Ratio Antibiotic/DNA

phosphorus

0.17-0.19

0.14

0.57-0.60

0.14

0.6 -0.625

* Supplied by the Upjohn Company, Kalamazoo,

Michigan.

fSupplied by the Warner-Lambert Research

Insti-tute, Morris Plains, N. J., research affiliate of Warner Chilcott Laboratories.

have a typical nucleic acid spectrum with

a peak at 260 ti.. Assay of these supernatants again showed distinct antibacterial activity

in all of the specimens examined.

Complex formation was next investigated

in systems containing antibiotic and highly

purified DNA, RNA, or polynucleotides of

varying chain lengths. To determine the

amount of antibiotic bound experiments

were carried out as follows. Increasing

amounts of antibiotic were added to a series

of tubes containing a constant amount of

DNA. The tubes were centrifuged at 13,000

RPM for one hour, and the optical density

of the supernatant solution measured at

260 &. In this manner the minimum amount

of antibiotic required to precipitate all the

DNA was determined, and from this value

the ratio of antibiotic to DNA-phosphorus calculated. Figure 2 shows the results of a

typical experiment carried out with calf

thymus DNA and neomycin. The curve is

linear and intersects the abscissa at a value

of 80 neomycin base. Table I presents

the results of experiments carried out with

other antibiotics. The data is presented as mole of antibiotic bound per mole of DNA

phosphorus. Table II lists several

antibi-otics which did not precipitate with DNA

in the test system.

Preparations of DNA isolated from thy-mus, liver, and spleen of calf demonstrated

identical precipitation curves with

neomy-cm, and were thus indistinguishable in the test system. The characteristics of the re-action were not altered by prior treatment of the DNA with heat.1#{176}A sample of DNA isolated from Ehrlich Ascites cells yielded

the same molar ratio of antibiotic to

DNA-phosphorus as found for DNA prepared

TABLE II

ANTIBIOTIcs NOT PRECIPITATED WITh DNA

Chloramphenicol*

Tetracycline

Methicillin

Penicillin G

Bacitracin

* Supplied by Parke, Davis, and Company, Detroit,

(4)

.2

-1.0

-08

-0.6

-0.4

-0.2

-0 ,0

c.,J

TUBE NO.

Fic. 3. Fractionation of polynucleotide fragments produced from calf thymus DNA by the formic

acid-diphenylamine reagent. Peak 1 = pCp; peak

2 = pTp; peak 12 = octonucleotides to

dodeca-nucleotides.

from the various tissues of the calf. It was of interest to examine the behavior of RNA

in the test system with neomycin, even

though RNA has not been detected by us

in human pulmonary 4 Both

yeast RNA and transfer RNA yielded molar

ratios of antibiotic to nucleic acid phos-phorus identical to that obtained with DNA

in the same system.

Since DNA and RNA exhibited identical precipitation curves with neomycin, it

be-came of interest to determine whether

chain length governed the precipitation

phenomenon. Figure 3 shows the elution

pattern of a sample of calf thymus DNA

after treatment with Burton’s reagent.7 The chain length of the pyrimidine

frag-ments varied from the monomers

pCp(5’-0-phosphoryldeoxycytidine 3’-phosphate)

and pTp(5’-O-phosphorylthymidine

3’-phosphate), eluted with 0.25 M ammonium

formate, to fragments with an average chain

length of 10, eluted with 2 M ammonium

formate. It was found that pTp and pCp

did not form precipitates with neomycin, nor did the polynucleotide chains in peaks

3 to 11.

Figure 4 shows the reaction of the

poly-nucleotides from peak 12 (eluted with 2 M

ammonium formate and having an average

chain length of 10) with neomycin. About two-thirds of the polynucleotide chains in this fraction are precipitated by neomycin,

0 80 60 240 340 420 500

pg NEOMYCIN SO4

FIG. 4. The precipitation of polynucleotides

corn-posed exclusively of prirnidine residues (average length = 10) by neomycin sulfate.

whereas the remainder are non-precipitable

even in the presence of an excess of the

antibiotic.

The digestion of calf thymus DNA by

streptodornase produces a spectrum of pro-ducts ranging in size from the monomer to

polynucleotides of a chain length of about 10.8 Figure 5 shows that about 50% of the

decanucleotide fraction eluted with 2 NI

ammonium formate buffer from a

Dowex-1-formate column was precipitated by

neo-mycin, while the remainder of the

poly-nucleotide chains in the fraction were

non-precipitable even in the presence of a

large excess of antibiotic.

The influence of ionic strength on

com-plex formation and precipitation of DNA

by neomycin was examined. The ionic

strength was adjusted by the addition of

sodium chloride to the reaction mixture. Figure 6 shows that at an ionic strength of 0.225 or less the added electrolyte had no

(5)

718

0

(0 c’J

0.4

-0.2

-50

00

80

60

40

20

I-z

I-z a::

w

a-c/I

z

z a

JLQ NEOMYCIN BASE

Fic. 6. The tirect of ionic strength on the precipi-tation of DNA 1) neomvcin sulfate. crosses =

water; open circles = ionic strength 0.225; half-shaded circles = ionic strength 0.250; closed

circles = ionic strength 0.3.

0 20 40 60 80

I.2#{149}

0.8

0.6

0 25 50 75 100 125

NEOMYCIN SO4

Fic. 5. The precipitation of polvnucleotides

(aver-age chain length = 10) obtained from a strepto-dornase digest of DNA by neornycin sulfate.

tile right, and at an ionic strength of 0.3 or

higher the marked effect of the added

dee-trolyte in the direction of dissociation of

the complex completely prevented precipi-tation of the DNA by neomycin.

It was of interest to sttidy the effect of

pH on the precipitation of DNA by

neo-mycin. The reaction was carried out in

dilute buffers

(

ionic strength = 0.1 or less)

of the desired pH. At pH values from 4 to

7, there was no detectable influence of

hydrogen ion concentration on the reaction.

At pH8, and higher values, precipitation of DNA by added neomycin was not observed.

COMMENT

In 1947, Cohen used streptomycin to

study some aspects of bacteriophage

sur-face structure. In this study, he observed

that the addition of streptomycin to neutral

solutions of high polymer DNA resulted in

the formation of opalescence or of floccu-lent precipitates.1 Cohen studied some of

the parameters of the reaction and showed

that whereas slight depolymerization of the DNA witil nuclease did not alter its pre-cipitability, alkali-degraded DNA or RNA

showed distinctly less precipitability with

the antibiotic. He also discovered that the complex could be dissociated in M sodium

chloride. In 1960, Cohen and Lichtenstein used streptomycin to isolate DNA and RNA from bacterial extracts. The DNA precipi-tates were found to contain 0.5 to 0.7 mole of streptomycin per mole of phosphorus.

The failure to demonstrate

neomycin-polymixin activity in the soluble fraction

of the 1% reconstituted secretions of patients with cystic fibrosis, in contrast to the easily demonstrable activity in less purulent secre-tions of patients with bronchiectasis and

laryngectomy, suggested that the higher

levels of DNA present in cystic fibrosis

secretions might account for the observed

phenomenon. That this was the case was

conclusively demonstrated by the liberation of the antibiotic from the insoluble fraction

(6)

with DNase I or by soltibilization of both

I)NA and antibiotic in NI NaCl.

The reaction of nticleic acids with

ileo-mycin has 1)een extensively studied. All

of tile samples of l)NA which were

cx-amined had extremely high molectilar

weights and were indistinguishable in the

reaction witil the antibiotic. Heat

treat-ment,1#{176} which presumably results in the conversion of DNA to the single-stranded form with a resultant llalving of tile

molec-ular weight, had no effect on the

cilaracter-istics of the precipitation reaction. Similarly,

two samples of RNA, of much lower molec-ular weight, yielded precipitates with the

same molar ratio of antibiotic to nucleic

acid-phosphorus as obtained with high

polymer DNA.

The preparation and isolation of pyrimi-dine polynucieotide fragments, and of poly-nucleotide fragments from a streptodornase

digest of DNA permitted a determination of tile minimum cilain length required for

precipitation to occur. The average chain

length of the precipitable fragments was

ten, and probably the range was from

octonucleotides to dodecanucleotides. Since

one-half to two-thirds of the polynucleotide

chains formed precipitates with neomycin,

it is concluded tilat tile critical chain length

is about 10. The fact that fragments

con-taming only pyrimidine nucleotide

pre-cipitate with neomycin strongly suggests that base composition and sequence are not important in the reaction.

The crucial clinical question is whether

the type of complexing described is of

significance at the site of infection.

Assum-ing there are about 100 gm wet weight of

secretions in the lung, the cystic fibrosis

secretions would contain 400 mg of DNA

capable of binding 100 mg of neomycin

suffate. The magnitude of the binding in

bronchiectasis secretions would be of the order of 40 mg of the same antibiotic. The

pH of both types of secretions varies from 6.8 to 7.2, and is entirely compatible with

complex formation. The experiments

car-ried out with reconstituted secretions

rep-resented five- to tenfold (hlutiOlls of \VilOie

secretions, and thus ionic strengtil was

clearly hot d factor. \Vllcn whole, fresilly

exl)ectOrated secretions froill 1)cttients vith

cystic fibrosis receiving an aerosol of

neo-mycin sulfate four times a day 1 were

ex-amined, both free and complexed antii)iotic

were detected. This finding suggests that

at least high in the tracheobronchial tree,

there was some degree of dissociation, or

that tile complexing ability of the DNA

had been exceeded. The chemical milieu at

the actual site of infection, determined by the degree of cellular necrosis, leucocytic

response, and bacterial action is difficult to

determine. However, it is likely that the

concentration of DNA is highest at that

site.

In consideration of the basic difficulty of

introducing antibiotics into the lung,

whether by systemic administration or

by aerosol, it would appear that the level

of DNA in the secretions might serve as an

effective barrier to therapy with basic anti-biotics. In order to overcome this barrier,

drainage to remove purtilent material, or

tile use of nucleases or salt solution to break

tile complex might be effective. It is evident

that increasing tile level of the antibiotic

to exceed tile complexing ability of the

DNA, or the use of antimicrobial agents

not complexed are also stlltal)le approaches

to the problem.

SUMMARY

1. Complex formation and precipitation

of DNA by neomycin, with consequent

in-activation of the antibiotic, has been shown

to occtir in the pulmonary secretions of

patients \Vitil cystic fibrosis.

2. Highly polymerized l)NA from a

variety of sources, as well as two samples

of RNA, were precipitated by neomycin in

vitro. Polymixin, kanamycin, colymycin,

and streptomycin similarly co-precipitate with DNA in vitro.

3. The minimum chain length of

poiy-nucleotide required for precipitation in the

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720

4. The complex is readily attacked by

DNase I resulting in the splitting of the DNA and the liberation of the antibiotic.

Both components of the complex are solubi-lized in NI sodium chloride.

5. The resistance of purulent foci of

in-fection to therapy with basic antimicrobial agents may, in part, be due to the complex

formation with the high levels of DNA

found at the site of infection.

REFERENCES

1. Cohen, S. S.: Streptomycin and desoxyribonu-clease in the study of variations in the prop-cIties of a bacterial virus.

J.

Biol. Chern.,

168:511, 1947.

2. Cohen, S. S., and Lichtenstein,

J.:

The isolation of deoxyribonucleic acid from bacterial

ex-tracts by precipitation with streptornycin.

J.

Biol. Chern., 235:PC55, 1960.

3. Potter, J. L., Matthews, L. W., Lernrn, J., and Spector, S.: Human pulmonary secretions in health and disease. Ann. N.Y. Acad. Sci.,

106:692, 1963.

4. Matthews, L. W., Spector, S., Lemm,

J.,

and Potter, J. L.: Studies on pulmonary secre-tions. I. The overall chemical composition of

pulmonary secretions from patients with

cystic fibrosis, bronchiectasis, and laryngec-tomy. Amer. Rev. Resp. Dis., 88: 199, 1963. 5. Kay, E. R. M., Simmons, N. S., and Dounce

A. L.: An improved preparation of sodium desoxyribonucleate. J. Amer. Chem. Soc.,

74:1724, 1952.

6. Kay, E. R. M., and Dounce A. L. : The

prepa-ration of sodium ribonucleate with the use

of sodium dodecyl sulfate.

J.

Amer. Chem.

Soc., 75:4041, 1953.

7. Burton, K., and Petersen, C. P. : The quantita-tive distribution of pyrimidine nucleotides in caff thymus deoxyribonucleic acid.

Bio-chem. Biophys. Acta (Amsterdam) 26: 667, 1957.

8. Felix, F., Potter, J. L., and Laskowski, M.:

Ac-tion of venom phosphodiesterase on deoxy-ribooligonucleotides carrying a

monoesteri-fled phosphate on carbon 3.

J.

Biol. Chem.,

235:1150, 1960.

9. Potter,

J.

L., and Laskowski, M.: Concerning the specfficit of streptococcal deoxyribonu-clease.

J.

Biol. Chem., 234: 1263, 1959.

10. Bollum, F.

J.

: Thermal conversion of

non-priming deoxyribonucleic acid to primer. J.

Biol. Chem., 234:2733, 1959.

11. Matthews, L. W., Doershuk, C. F., Wise, M.,

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1965;36;714

Pediatrics

Joseph L. Potter, LeRoy W. Matthews, Samuel Spector and Joy Lemm

DEOXYRIBONUCLEIC ACID IN HUMAN PULMONARY SECRETIONS