JOURNAL OF BACIERIOLOGY, Feb., 1966 Vol. 91, No. 2Copyright © 1966 American Society for Microbiology Printed In U.S.A.

Cationic Proteins of PolymorphonuclearLeukocyte Lysosomes

I. Resolution of Antibacterial and Enzymatic ActivitiesH. I. ZEYA AND J. K. SPITZNAGEL1

Department of Bacteriology and Immunology, University ofNorth Carolina School ofMedicine,Chapel Hill, North Carolina

Received for publication 11 October 1965

ABSTRACT

ZEYA, H. I. (University of North Carolina, Chapel Hill), AND J. K. SPITZNAGEL.Cationic proteins of polymorphonuclear leukocyte lysosomes. I. Resolution ofantibacterial and enzymatic activities. J. Bacteriol. 91:750-754. 1966.-A lysosomalfraction from polymorphonuclear (PMN) leukocytes of guinea pig peritoneal exu-date was subjected directly to electrophoresis on cellulose acetate paper treatedwith cetyltrimethyl ammonium bromide. The lysosomal components resolved intoseven bands moving towards the cathode. Assay of the eluted bands showed that theantibacterial activity was distinct from lysosomal enzymes and was associated withthree cationic components (bands I, II, and III) which migrated most rapidly to-wards the cathode, ahead of lysozyme ribonuclease and deoxyribonuclease. Qualita-tively, the antibacterial components appeared to be rich in arginine. The antibac-terial components were absent in the pherograms of nuclear fractions of PMN leu-kocytes and in supernatant fractions that remained after lysosomes were removedfrom cell homogenates by centrifugation at 8,000 X g.

Leukocytic degranulation and participation ofpolymorphonuclear (PMN) granules in the bac-tericidal activity of leukocytes were described asearly as 1894 by Kanthak and Hardy (6). Overthe past decades, a number of partially charac-terized antibacterial substances in extracts ofwhole PMN leukocytes have been reported byvarious workers (10). As nuclei of PMN leuko-cytes, like nuclei of all mammalian cells, containhistones and protamines that in themselves pos-sess antibacterial activity (10), the antibacterialproperty of whole leukocyte extracts could notdefinitely be attributed to extranuclear compo-nents. Utilizing cell fractionation methods, Cohnand Hirsch (2) demonstrated a number of acidhydrolases, as well as the bulk of the leukocyticantibacterial activity, in association with specificgranules of PMN leukocytes. As these granulescontained acid-soluble enzymes, such as lysozyme,ribonuclease, and deoxyribonuclease, the natureand the existence of the antibacterial substancesindependent of the hydrolytic enzymes could notbe defined. The indication that the substances in-volved may be polycationic in nature came withthe histochemical studies of Spitznagel and Chi(11).

1 Publc Health Service Research Career Develop-ment Awardee.

Using a method by which specific granules ofPMN leukocytes can be subjected directly to elec-trophoresis, we have separated the contents ofgranules into several components which include,apart from known enzymes, three unique basicprotein components possessing antibacterial ac-tivity. A preliminary report on some aspects ofthese studies has been published elsewhere (13).

This paper and the one following it describe theseparation and the biochemical and biologicalcharacterization of cationic proteins isolated fromspecific granules of PMN leukocytes.

MATERIALS AND METHODSPMN leukocytes. The leukocytes were obtained

from guinea pig peritoneal exudate induced by injec-tion of 0.5% glycogen in 0.85% saline. White maleguinea pigs weighing 400 to 600 g were used. The aver-age peritoneal yield (80 to 100 ml) contained approxi-mately 5.0 X 106 cells per milliliter. Of these cells,95% were PMN leukocytes.

Preparation of granule fraction. The granules fromPMN leukocytes were prepared by homogenizationand differential centrifugation of disrupted cells in0.35 M sucrose. The granular fraction (8,000 X g) wassuspended in 0.25 M sucrose and stored at 4 C. Thenuclear fraction (twice washed with 0.25 M sucrose toremove contamination with large granules) and thepost granular supernatant fraction were saved forelectrophoretic comparison.

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LYSOSOMAL CATIONIC PROTEINS. I

Leukocytes from peripheral blood. The leukocytesfrom peripheral blood of the guinea pig were separatedaccording to the technique of Athens et al. (1). Thered blood cells were removed by dextran agglutinationand hemolysis by 5% dextrose.

Paper electrophoresis of granules. Oxoid paper(20 by 5 cm; Colab Laboratories, Inc., ChicagoHeights, Ill.) was soaked in 0.5% cetyltrimethyl am-monium bromide (CTAB) in water for 3 min and wasrinsed with acetate buffer (pH 4) for 5 min. Samplesof granule suspension in 0.25 M sucrose were appliedto strips from drawn tips of Pasteur pipettes. Electro-phoresis was carried out for 1 hr (200 v; 0.002 amp)at room temperature in acetate buffer (pH 4; ionicstrength, 0.05). The electrophoretic strips were stainedwith 0.5% Buffalo Black (Allied Chemical Corp.,Morristown, N.J.) in methanol with 10% (v/v) glacialacetic, and were rinsed with a 10% solution of glacialacetic acid in methanol.

Elution of biological activity from pherograms. Thelong edge of an unstained pherogram was cut away,stained with Amido Black, and used as a marker forelution of individual bands from the remainder. Forantibacterial assay, protein was eluted from individualbands with 0.5 ml of 0.01 N HCI. Elution was carriedout directly in 1-cm dialysis tubing for 30 min. There-after, the paper strip was removed from the bag tominimize loss of material by adsorption, and theeluate was dialyzed against 0.01 N HCI (2 liters) for 6hr, under cold temperature, to remove the residualCTAB.

Antibacterial assay. The assay was carried out ac-cording to the method of Hirsch (4) in 2-ml plasticcups (Linbro Chemical Co., New Haven, Conn.). Acitrate phosphate buffer of pH 5.6 was used as amedium for assay. Throughout the experiments, 18-hrcultures of Escherichia coli strain 0117:H27 wereemployed. Fifty per cent inhibition of bacterial growthas estimated by comparison with control cups wastaken as the end point.

Lysozyme assay. Lysozyme activity in each bandwas determined by the procedure of Shugar (9).Lysozyme substrate consisting of ultraviolet-irradiatedMicrococcus lysodeikticus was obtained from Difco.Tests were standardized with crystalline egg whitelysozyme. Individual bands from electrophoreticstrips were eluted in 1.5 ml of 0.1 M phosphate buffer(pH 6.2) for 30 min, and eluates were assayed forlysis of M. lysodeikticus.

Ribonuclease assay. Ribonuclease was assayed ac-cording to the turbidimetric method of Houck (5).Commercial yeast ribonucleic acid (RNA) was usedas the substrate. Elution of individual bands was car-ried out in 1 ml of 0.1 M acetate buffer (pH 5) for 30min. The turbidity was measured at 400 mt& in a Beck-man DB spectrophotometer.

Deoxyribonuclease assay. Deoxyribonuclease ac-tivity was determined according to the method ofSchneider and Hogeboom (8). Highly polymerizedDNA (Calbiochem) was used as substrate. Individualbands from electrophoretic strips were eluted in 1 mlof 0.1 M acetate buffer (pH 5) for 30 min. The eluateswere incubated with the substrate for 2 hr at 37 C.The amount of deoxyribonuclease activity in each

band was determined by comparison with the standardcurve.

RESULTS

Resolution of PMN granule proteins Attemptsto resolve the constituents of PMN granules byzone electrophoresis on untreated acetate paperin a pH range from 2 to 9 were unsuccessful. Thesubstances remained at the line of application,and even at very acidic pH failed to separate intobands. It was also difficult to elute the materialfrom the paper. To avoid electrostatic bond for-mation between cationic groups in the proteinsand the carboxyl groups of the cellulose acetate,the paper was treated with CTAB, a cationic de-tergent. The presence of residual CTAB on thepaper and the acid pH of the suspending bufferseemed to cause the immediate lysis of the affixedgranule suspension. Subsequent electrophoresison the neutralized paper resolved granule compo-nents into seven or more bands, which migratedtoward the cathode as shown in Fig. 1. The pat-tern was found to be reproducible in experimentsfrom day to day.

For the purpose of description, the bands weredesignated by Roman numerals according to theirmobilities towards the cathode, bands I and IIbeing the fastest-moving components. The per-centage of various electrophoretically separatedcomponents of PMN granules was estimated bydetermination of bound dye, on the assumptionthat bound dye would be proportional to thequantities of protein in the stained bands and thatintrinsic dye-binding capacity of the componentswould be equal. The results are summarized inTable 1.Bands I and II possessed the fastest mobilities

and were found to be variable. At times, theyseparated into more than two bands or a singletrailing fast-moving band. Band III was stainedmore heavily than the other bands, with a charac-teristic deep-blue color, providing an easily rec-ognizable and very reproducible reference pointin these patterns.

Biological activities associated with protein

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FIG. 1. Zone electrophoresis of PMN lysosomes.Antibacterial bands: I, II, and III. Lysozyme: IV.Ribonuclease: V. Deoxyribonuclease: VI.

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ZEYA AND SPITZNAGEL

TABLE 1. Estimation of relative protein content and biological activities of variouscomponents obtained by electrophoresis ofPMN lysosomes

Band Mobilitya Relative protein Antibacterial Lysozyed Ribonucleased Deoxyribo-Contentb activity" yoynd Rbnces nucleased

I + II 0.65 10 16 0 0 0III 0.53 24 32 0 0 0IVe 0.40 18 0 0.63 0 0Vs 0.29 20 0 0 0.5 0VIP 0.14 3 0 0 0 0.2VII 0.10 4 0 0 0 0Origin 0 21 0 0 0 0

a Microns (at pH 4) cm'hr'lv'1.b Relative percentage of Amido Black per band.c Reciprocal of highest dilution showing 50% growth inhibition.d Micrograms of crystalline egg white lysozyme, pancreatic ribonuclease, pancreatic deoxyribonu-

clease equivalent.* Samples of purified commercial lysozyme, ribonuclease, and deoxyribonuclease displayed mobilities

in this system identical to bands IV, V, and VI, respectively.

bands. For various assays, materials from individ-ual bands were easily eluted. The antibacterialassay of bands eluted from electrophoretic stripsrevealed that the activity against E. coli was con-fined to bands I, II, and III, which migrated mostrapidly towards the cathode (Fig. 1). Because oftheir low protein content, bands I and II wereassayed together. The antibacterial activity isshown in Table 1 as the reciprocal of highest dilu-tion showing 50% growth inhibition as comparedwith the controls. Usually, complete inhibitionwas found in cups preceding the 50% endpoint.Assay for lysozyme showed the activity exclu-

sively in band IV (Fig. 1; Table 1). Ribonucleasewas detectable only in band V, whereas band VIshowed deoxyribonuclease activity. Under theseconditions, the arrangement of enzymatic bandsseemed to follow closely their isoelectric points.Samples of purified commercial lysozyme, ri-

bonuclease, and deoxyribonuclease displayedmobilities identical to bands IV, V, and VI, re-spectively (Fig. 2).A qualitatively identical pattern was obtained

with granules from peripherally circulating PMN.Histochemical properties ofbands resolvedfrom

PMNgranules. Histochemical techniques (7) werealso employed for the detection of arginine-richcompounds and carbohydrate- and lipid-contain-ing substances in the electrophoretically separatedcomponents of PMN granules. The strips werestained with a Sakaguchi method that revealedthe presence of high arginine content in bands I,II, and III. The electrophoretic strip was found tobe periodic acid-Schiff and Sudan black negativeexcept at the point of application, indicating thatunder these conditions the compounds containingcarbohydrate and lipid remained at the origin.

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FIG. 2. Comparative pherogram of samples ofpuri-fied commercial lysozyme (Lys.), ribonuclease (RNA-ase), deoxyribonuclease (DNA-ase), and PMN lyso-somes (GRN.). Antibacterial bands: I, II, and III.Lysozyme: IV. RNA-ase: V. DNA-ase:VL

Comparison ofpherograms of granular, nuclearand post granular supernatant fractions. Samplesfrom the supernatant fraction and the nuclearfraction of PMN leukocytes were com edelectrophoretically with granule fractions. Theresults (Fig. 3a, 3b) revealed marked differ-ences in the electrophoretic patterns of the differ-ent fractions. The nuclear fraction did not seemto resolve into definite bands, and the bulk of thematerial stayed at the point of application. In thispreparation, care was taken to remove the con-taminating large granules by repeated washings ofthe fraction with 0.25 M sucrose.

In the supernatant fraction, some pattern wasdiscernible, although quite different from the

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LYSOSOMAL CATIONIC PROTEINS. I

FIG. 3a. Comparative pherogram of supernatant

fraction after centrifugation at 8,000 g X (SUP.) and

PMN lysosomes (GRN.). Antibacterial bands: I, 11,

and III. Lysozyme: IV. RNA-ase: V. DNA-ase: VI.

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FIG 3b. Comparative pherogram of nuclear frac-tion (NUC.) and PMN lysosomes (GRN.) Antibac-terial bands: I, II, and III. Lsyozyme: IV. RNA-ase: V.DNA-ase: VI.

granule fraction. Bands corresponding to I, II,

and III of intact granules were absent in the pher-

ogram of the supernatant fraction. However,some smudged, streaked material was visible at

the level of bands I, II, and III of the granulefraction. Another band corresponding to the

ribonuclease band of intact granules was visible

in the supernatant fraction. These substances

either could have been derived from some rup-tured granules during the process of fractionationor they may have represented components origi-nating from ribosomes ofPMN cytoplasm. How-ever, a consistently reproducible electrophoreticpattern ofPMN granules quite different from thatof nuclear and supernatant fractions showed thatthe components visible on the granule pherogramseemed to have their origin in PMN granules.The fact that the granule fraction could be di-

rectly subjected to electrophoresis was a definiteadvantage over extraction procedures wherebysome important components might escape anal-ysis or be altered.

DIscussIoN

The use of paper electrophoresis in the separa-tion of cationic proteins has been severely limiteddue to the avidity with which cationic proteins are

adsorbed to materials such as cellulose acetate.The paper acquires a negative charge when incontact with buffer solutions, thereby leading toadsorption of the positively charged proteins tothe paper (12). Under these conditions severestreaking has occurred with the result that theresolution of such protein mixtures has been un-satisfactory. To minimize the electrostatic adsorp-tion of cationic proteins to paper, attempts havebeen made to esterify the carboxyl groups of thepaper with diazomethane (12), or to block themwith detergents such as sodium cetyl-sulfonateand Tween 20 (3). By these methods, a positivelycharged paper is produced on which cationic pro-teins can move below their isoelectric points withlittle or no adsorption. Proteins bearing net nega-tive charge are adsorbed on such modified paper.

In our studies, pretreatment of the paper withthe cationic detergent CTAB made possible thedetermination of mobilities of lysosomal cationicproteins and the use of these mobilities as ananalytical tool. The residual CTAB on the paperand the acidity of the buffer helped in immediatelysis of applied granules. This offered the advan-tage of direct electrophoretic analysis of the sub-cellular components of PMN. The pherogramswere consistently reproducible. The detergent-treated paper did not seem to exert a detrimentaleffect on the biological activities of the proteincomponents. In this respect, the short runningtime of 1 hr required for electrophoresis was un-doubtedly advantageous.The electrophoretic pattern showed that basic

proteins possessing diverse biological activitiesresolved under these conditions. The granule com-ponents resolved into multiple bands of which I,II, and III were found to be antibacterial, andbands IV, V, and VI accounted for lysozyme, ri-bonuclease, and deoxyribonuclease activities,respectively. The mobilities toward the cathodeseemed to be directly proportional to the isoelec-tric points of the known proteins.The antibacterial activity of PMN granules was

demonstrable only in the fastest-moving bands, I,II, and III. At pH 4, this fast mobility did notnecessarily mean that proteins in these bands werebasic, but the fact that they moved faster thanlysozyme pointed to the possibility of their havingisoelectric points above 11.0. The histochemicaldetection of high arginine content in these bandswas yet another indication of the basicity. Furtherevidence of the strongly cationic nature of theproteins in bands I, II, and III of PMN lysosomesis discussed in the following paper.

Nuclear basic proteins like histones and prota-mines are known to have antibacterial effects,

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ZEYA AND SPITZNAGEL

and PMN leukocyte nuclei, like all mammaliancells, contain histones. The possibility of nuclearcontamination of the granular fraction (8,000X g) was minimized by careful homogenizationof leukocytes in hypertonic sucrose solution inthe cold, followed by repeated sucrose washingof the fraction. Consistently reproducible differ-ences in pherograms of granules compared withthose of nuclear fraction showed that the compo-nents essentially originated from PMN granules.A qualitatively identical electrophoretic patterngiven by PMN lysosomes of peripheral bloodwould indicate that the antibacterial proteins donot arise from the inflammatory conditions pro-duced during the induction of aseptic peritonealexudation.A comparative study, with the help of this

technique, of basic protein composition of PMNlysosomes may provide a useful area for the inves-tigation of species- and disease-associated varia-bility of resistance against infection.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService grants GMK3-15-155 from the Division ofGeneral Medical Sciences and AI-02430 from theNational Institute of Allergy and Infectious Diseasesand by General Research Support grant 1-GS-106.We gratefully acknowledge the expert technical help

of Marjorie H. Cooney.

LITERATURE CrrED

1. ATHENS, J. W., A. M. MAUR, H. ASHENBRUCKER,G. E. CARTWRIGHT, AND M. N. WINTROBE.1956. Leukokinetic studies. A method for label-

ing leukocytes with DFP. J. Haematol. 14:303-331.

2. CoHN, Z. A., AND J. G. HnISCH. 1960. The isola-tion and properties of specific cytoplasmicgranules of rabbit polymorphonuclear leuko-cytes. J. Exptl. Med. 112:983-1009.

3. FLODIN, P., AND F. J. LOOMEIJER. 1953. Zoneelectrophoresis. Advan. Protein Chem. 8:461-468.

4. HIRSCH, J. G. 1958. Bacteridical actions of his-tones. J. Exptl. Med. 108.924-944.

5. HoucK, J. C. 1958. The microdetermination ofribonuclease. Arch. Biochem. Biophys. 73:384-386.

6. KANTEAK, A. A., AND W. B. HARDY. 1894. Themorphology and distribution of the wanderingcells of mammalia. J. Physiol. 17:81-119.

7. PEAusn, A. G. C. 1960. Histochemistry: theoreticaland applied. Little, Brown & Co., Boston.

8. SCHNEIDER, W. G., AN G. H. HoGErmoM. 1952.Intracellular distribution of enzymes, deoxy-ribonuclease, and ribonuclease. J. Biol. Chem.198:155-163.

9. SHUGAR, D. 1952. The measurement of lysozymeactivity and the ultraviolet inactivation of lyso-zyme. Biochim. Biophys. Acta 8:302-309.

10. SKARNES, R. C., AND D. W. WATSON. 1957. Anti-microbial factors of normal tissues and fluids.Bacteriol. Rev. 21:273-294.

11. SPITZNAGEL, J. K., AND H. Y. Cm. 1963. Cationicproteins and antibacterial properties of infectedtissues and leukocytes. Am. J. Pathol. 43:692-711.

12. TISELIUS, A., AND P. FLODIN. 1953. Zone electro-phoresis. Advan. Protein Chem. 8:461-468.

13. ZEYA, H. I., AND J. K. SPITZNAGEL. 1963. Anti-bacterial and enzymic basic proteins from leu-kocyte lysosomes: Separation and identifica-tion. Science 142:1085-1087.

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