Histamine binding by heparin

ARC’HIVES

OF

BIOCHEMISTRY

ASI)

BIOPHYSICS

Histamine

96,

20-27

Binding

YUTAKA

Recei\-ed

(1962)

by

Heparin’

KOBAYASHI

April

6, 1961

Histamine was bound strongly by heparin in 1% acetic acid, distilled water, or 0.01 N HCl in the absence of cations. The histamine-heparin complex was unstable in salt solutions. Polyamines, in equimolar concentration as histamine, int,erfered with the ability of heparin to bind histamine. Histamine was bound selectively by heparin in the presence of equimolar amounts of cadaverine, scrotonin, and adrenaline. Desulfated heparin failed to bind any histamine. Proteins containing a high percentage of glutamic and aspartic acids showed little histamine-binding activity. A hypothesis for the histaminebinding mechanism of the mast cell and the release of histamine by Compound 48-80 is presented.

which may help rationalize the results of others in the field and presents a hypothesis concerning histamine binding by mast cells based on the data presented.

INTRODUCTION

The relationship between heparin and hi&amine has been a subject of much intcrest since the discovery of the mast cells as a major depot of histamine in animal tissue (1). Many workers (2) have shown that heparin can bind histamine, but the affinity between heparin and histamine was found to be small. ilIacInto& (2) considered it unlikely that heparin as such could bind the quantity of histamine associated with the mast cells. Macintosh postulat’ed that if heparin were the binding agent in the mast cells, the native heparin of these cells must be more “histaminophilic” than commercial heparin or t,hat the mast cell milieu is peculiarly favorable to histamine binding by heparin. On the other hand, Amann and \?l:clrle (3, 4’1 have described a strong binding of histamine by heparin, in 1% acetic acid, and the displacement of histamine from the histamine-heparin complex by polyamincs. This paper describes dialysis t~xpcriincnts using radioactive histamine

EXPERIMENTAL Histamine, labeled with Cl1 in the 2 position of the imidazole ring, was obtained from the Nuclear Chicago Instrument and Chemical Corporation. Caduvarine, labeled with C” in the 1 or 5 position, was synthesized by Schaycr et al. (5). Sodium hepminate, 100 units/mg. was obtained from the Nutritional Biochemicals Corporation. The desulfated heparin was a gift from Dr. Floyd C. McTntirc of the Abbott Laboratories, North Chicago, Ill. Compound 48-80 was a gift from the Wellcome Research Laborat,orics, Tuckahoe 7, N. Y. Isotopic assays were performed in Packard Tricarb Liquid Scintillation Spectrometers, Models 314 and 314X. Thr assay solution consisted of 0.10 ml. sample, 0.10 ml. of 0.1 M phosphate buffer, pH 7.5, 3.0 ml. of nbsolutc alcohol, 7.0 ml. toluene, and 30 mg. diphenylosazole. The solution was counted in spcv*inl low-potassium 5-dram vials manufacl,uretl by the \Vheaton Glass Company. The counter was ol)crn(ctl at approximately 30% eficiency with a bnc~kground I)c$w?f,n 9 and 12 c.olmis/min. On(y microgram of lahcletl histamine nsbayrd :~pproxim:rirly 28,000 count.q/min. and 1 pg. of lab&d catlnverine assayed approximately 3000 c*ount.*:/min. under these conditions Thcx grnrral rsperimental procedure was as follows: tllc IrPparin or protein was c*ont:rinetl in

1 Thk investigation was supported br a research grant E-1135 from the National Instjtute of Allcl.gy anti Infectious Diseases, U. S. Public Health Scrvivc. National In&kites of Health and Contracat ‘\T(30-l)-2085 with the TJ. S. Atomic Energy Commiskon. 20

HISTAMINE

BINDING

1 ml, water in a IA-in. size V&king Nojax casing hag. The bag was then dialyzed against three changes of the dialyzate used for the experiment for :I minimum of 18 hr. of 4°C. The bag was then removed from the dialyzate, quickly blotted dry with adsorbent tissue paper, and placed into t,he test tlialyzate rontaining histamine-C’” and other additions (when used). At the end of the experimrnl, two O.I-ml. nlicluots were taken from the tlialysis sack and the dialyzate for analysis using I hc liquid scintillation spectrometer. The percent histamine bound was calculated on the basis of t,hc radioactivity found within the dialysis bag less the radio&i\-ity per milliliter found in the tlialyzate divided by the total amount of ratlio:tct,ivit,p found in bot,h the bag and the dialyzate ~1, the end of t,he experiment. When the stability of the histnminc-hrparin complex was studied, the bag containing the heparin was allowed to equilibrate with histamine-C4 for 48 hr. at 4”. The bag was then removed, blotted, and placed into 19 ml. of a fresh dialyzate. dt appropriate intervals, O.lO-ml. aliquots of the fresh dialyznte ll-cre nsayed to measure the efflux of histamine-C4 from the romplex into the dialyzate. Ascending paper chromatograms were run on :%-in. filter-paper strips (Hengar filter-paper roll) using a solvent system consisting of 50 parts etlr:mol, 40 parts water, and 1 part glacial acetic acid. The chromatogmms wrre run approximately

0

200

400 MICROGRAM

600 HISTAMINE

BP

21

HEPARIN

16 hr. Toluidine blue spray, 0.005%, gave a purple spot against. a blue background for heparin and the histamine-heparin complex but did not react with histamine. Pauly reagent spray was used to locate the position of histamine. Histamine gave :L red color against a yellow background. When both heparin and histamine were to be located, the c~hromatogram was first sprayed uTith toluidine blue, dried, and then sprayed with the Pauly reagent. The reverse procedure will result in t,he innbilit,y to loc:~te the position of heparin. The chromatograms were alao scanned for radioactivity using a Nuclear Chicago Actigraph with a model D-47 thin-window gas-flow counter. Demineralized water was prepared by passing distillr,d lv:lter through a column charged with a mixturf% of cationic,ant1 nniclnic~-c,xcllange resins. RESULTS HIRT.~MIR’E

Brsnmc,

BY HEPARIN

Factors involved in the binding of histamine by heparin mere studied. Figure 1 shows the effect of various concentrations of histamine in the diffusate on the amount of histamine bound by hepnrin for various concentrations of heparin. As one can see, beta-em the hepwrin concentrations of l-4 mg., a plateau was obtained around IOOO-

800 IN

1000 ISML

OF

5000 DIFFUSATE

FIG. 1. Histamine binding by heparin using varying concentrations of histamine in the tliffusnte. A 1 mg. hrpnrin/ml. in bag. B 2 mg. hcparin/ml. in bag. C 4 mp. hrpnrin/ml. in bag, Condit,ions: As described in trxt,. Dialysis time lvas 20-24 hr. Each v:~lup is an average of four determinations. The nhspissa rrpresents l,he original concentration of histamine in the diffusate.

5000 pg. of original histamine concentration. In a second experiment, the maximal histamine-binding capacity of varying amounts of hcparin was determined, and the results are shown in Fig. 2. The sulfate crluivalcnt of heparin WE calculated on the lrasis of a molecular weight, of 12,000 and a sulfwtc equivalent of 22 moles per molr of hcparin ( 4 1. In this experiment, a great deal of xcattrr of the yalucs was observed. Howseemed to exist ever, a linear relationship hct~wctw the concentration of lieparin and the total amount of histamine bound at’ saturating concentrations of histaniinc. It was found that histamine can he bound hy hcln~rin in 1% acetic acid as reported by Aniann and Werlc (3 1. It n-as also found that hq)arin will bind histamine in 0.01 S

HCl. \Vlwn a small amount of histamine (2 pg. 1 was used with 4 nig. lieparin, the hcparin bound l)etw-cen 60 and 90% of the histamine introduced in either the acetic acid or 0.01 S HCl dialpzatc.

A sample of liistalliinc-licl~~~rin complex was ol)tainetl hy placing a l-ml. dialysis sack containing I mg. heparin/ml. into a lwakcr containing 500 pg. of nonisotopic histaininc and 2 pg. histamine-Cl-l. After dialysis, it was found t)liat 430/O of the liistan& TWS hound. Then 0.10 ml. of the coml~lcx solut,ion was chromatographed as dcscrihcd under Methods. The chromatogram sl~owcd a radioactive peak at the hist,aminc position and a large peak at, tlw hcp-

HISTAMINE

BINDING

arin position (Fig. 38 j. When the chromatogram was sprayed with the Pauly reagent, a large clear spot appeared at the origin and a smaller diffuse spot appeared at the histamine posit,ion. There was a definite smearing of the red color increasing in intensity from the heparin position t,o the free histanline position with the Pauly ITagent (Fig. 3R). This tailing of hist’amine may br a reflection of the equilibrium bctwcn free histamine and the complex. As tllc free histaininr migrates :irTay from the coml)lex, sonic dissociut,ion of the complex must occur releasing free histamine. Since the equilibrium is heavily in favor of the complex, the amount of free histamine relcasccl must’ bccomc infinitesimal as the cllronlatogruphy procceda. When the chromat’ograms of either the complex or heparin alone were sprayed mit,h toluicline blue, a c~lcar single spot at the origin appeared showing that the heparin was almost st,ationary in the solvent used (Figs. 31) and 3E). The average range of Rf values for the liiataitiine-2icparin complex, heparin, and fret histamine were o-0.12, o-0.11, and 0.59-0.71 for a minimulti of ten determinat,ionu. Further evidence for the existence of a histamine-hcparin complex was obtained from the following expcrimcnt. One hundred micrograms of heparin vas placed on a paper strip 2 in. above the origin. One inicrogram of histan~inc-C14 was placed at the origin and allowctl t,o migrate in the solrent system used abow. This system was such that if no con~plex formation occurred between histamine and heparin, histamine would migrate through the heparin position since hcparin is stationary in the solvent system used. However, if complex formatlon occurred, then one would expect to find histamine at, the heparin position. It was found that virtually all the radioactivity wa5 at the hrparin position. STABILITY

OF WE HTSTANISE-HEPARIS COMPLEX

TVhcn a dialysis sack containing llistamine-Cl”-heparin comples was place{1 in :i fresh tlialyzatc of cit,hcr 176 a&c acid 01 w:tter, Iittlf~ to none of the radioactivity was

BY

HEPARIN

23

FIG. 3. I’aywr c.llrorn;~tog~n~~~~~~ of histaminehey:trin complt~. A. Ratfionctive scan of histamineC’“-heparin complex. Liquid scintillation counting data. indicntrtl 437 binding of histamine by heparin (xc test 1. 11. A\l)ovc chrom:ttogram st)Iqyrtl with Pnuly re:lgf‘nt. C. 5 pg. noniPolopic histamine :tlone ~pr:~ycd xvit,tl l’nuly reagent. D. Chromntogram il spr:tyed with tolnidine blue. E. 20 pg. hcp:il,in alonc~ >pr:iyfd Jvitlr toluiclinr blue.

found in the dialyzatc after a period of several hours. In water, the complex was st’able over a period of 72 hr. However, when a similar sack containing radioactive complex was placed in Tyrode’s solution, there was a rapid appearance of histamineCl’ in the dialyzate. Within 4 hr., the concentration of hist.aminc on either side of t,he nwmbrnne TWS almost equal; after 24 hr., the histuininc concentration was equal. ox HISTAMINE BISDING The instability of the histamine-hcparin complex in Tyrodc’s solution suggested t,hat rations int,crfere with the binding process. The effect, of increasing SaCI concentration (Table I J and of various cations at equal ionic strength (Table II J on the binding lnwcss were examined. The data show that cations do interferc with t,hc abilit,y of hcpwin to bind hist:wG~c. The multivalent ca-

~XFFECT

OF

CATIOM

24

IiOBAT.\SHI

tione were t~lie most effective inhibitors of the binding process. Sinw 0.01 -Y ?yTaCl had lit,tle effect on the bintling of a minute amount, of histamine by hqwin t Tables I and II), it was dcsircacl to see whether or not sodium ions would rompctc nit.11 liistaminc for heparin TIZRLE EFFECT

NnCl

OF

BY

ON

HEI’ARIN

IN

I HISTAMINE

BINI)ING

DISTILLED

WATER

Conditions: 2 pg. histamine-Cl4 fnsxte. Heparin concentration Time was 18 hr. Concn. sac1

Per cent

in was

histamine

It was of intorest to test the specificity of histamine binding by heparin. Histamine (3 pmoles) containing 4 pg. histamine-Cl4 was placed in a beaker containing 19 ml. of demineralized water and a dialysis sack with 1 mg. heparin in 1 ml. water. Eyuimolar amounts of spermine, spermidine, cadavcrine, serotonin, and L-adrenaline were added individually and the amount of histamine-Cl4 bound by hcparin was measurcd. Sperminc. spermidinc, serotonin, and L-adrenaline n-we also tested in equivalent basic concentrations as 3 ~molcs histamine in 19 ml. diffuswte. Compound 48-80 was tested at t,hrec levels: 550, 1000, and 2000 pg. in 19 ml. water. The rcsult,s are shown in Table IV. It can be seen that spermine, spermidine, and the highest concentration of Compound 48-80 tested prevented the binding of any histamine by heparin. However, at’ the conccntrat,ion tested, cadavcrinc, scrotoniq and L-adrenaline had little influence on histamine binding.

19 ml. dif1 mg./ml.

bound

.w

i2, 88 Av. 70, 73 Av. 7, 9 Av. 0, 2 Av.

0

0.01 0.1 1.0

TABLE I~FFECT

OF

VaRIor-s

STRENGTH HEPARIN

AT

HISTAMISE IN

DISTILLED

COIlCll.

E~rr.ir,

IONIC.

BISUING

BY

WATER

Conditions: 2 pg. hist,amine-Cl” diffusate. Heparin concentration Time was 18 hr. Salt

80 72 8 1

II

SAI,TS

ON

in 19 ml. of was 4 mg./ml.

Per cent

histamine

bound .____

.$I Co111

NaCl KC1 MgCI MIlCl~ C&l FeCl AlCl

I.01

S :’ a :j

i

0.01 0.01 0.0033 0 ,003:~ 0.0033 0.00166 0.00166

61. 70, 6-1. 13, 8, 8, 11, i 0.

69, 72, 88 Av. 73 Av. 76, 82 .4v. li Av. 17 ;Iv. 12 Av. 21 Av. 2 Av.

75 72 74 15 13 10 19 1

Since cadaverine in the presence of an eyuimolar amount of histamine was not bound by heparin, it became of int’ercst to dcterminc whether or not hcparin would

TABLE THE

Conditions:

HISTAMINE-BINUING

As described

in teat.

I--;-rug.

heparin

pg. bound/

0.86, 0.99, Av.

1.1 1.3

= 1.01

OF

Heparin

102

0.95, 0.91,

III

CAPACITY

Micrograms

Histamine,

at increased histamine concentrations. Therefore, the specific binding capacity of 1 mg. hcparin for hi&mine n-as determined in 0.01 ,I’ n’aC1 (Table III). Uy comparison with tlat,:r in Fig. 1, it can be seen that the specific binding capacity w:w wtluced, but that which rcwiainctl could still bind :L rclatively lurgc :mwunt~ of 1iist:m~ine.

histamine-CIA

= 40.8

0.01 5 iY;nCl was 1 mg./ml.

IN

in 10 ml. of iliffusate

504

I

29, 38, 10 40, 46, 52 Av.

HE~ARIX

concentration

68, 94, 95 103, 107, 111 ~

Av.

= 96

I

1004

71, 106, 110 1 120. 122, 176

I 1

Av.

= 118

5004

I

~ 125, 150, 155 ( 175, 185, 190 Av.

= 163

HISTAMIKK

BI?;DISG

BT

TABLE EFFECT

OF OTHER

AMISES

IV

Ox HISTAMINE

Conditions:

BINDISC:

As described

RP HEPARIN

in text I

Concn.‘~

Substance

25

HEP.~RI~

Per cent

histamine

bound

by 1 mg. heparin

IAverage

~vulles!70

Histamine-Cl4

alone

Histamine-Cl4 Histamine-Cl4 Histamine-C” Histamine-Cl4 Histamine-Cl4 Histamine-Cl4 Histamine-Cl4 Histamine-Cl4 Histamine-Cl4 Histaminc-C*4

plus plus plus plus plus plus plus plus plus plus

IIist,amine-Cl4

plus

Histamine-C”

plus

‘A Concentration

ml.

other

37, 40. 35, 44 35: %!I,

40, 47. 42, 52, 42. 30.

43. 52, 48, 67 43, 30,

jq.

homtl/

~

~

44 31, 33, 3i 21

16, 21. 23. 23. 24 0. 0

than

0

llistaminc~.

12. 13 14, 14 Av.

= 13

91, 102 IOT.

Av.

bincl cadaverine in t’he absence of any othei amine. Therefore, the specific binding capacity for cadaverine of 1 mg. heparin was determined using cadaverine-Cl4 under the same conditions as for histamine (Ta,ble I). The results, prescnt,etl in Table Y, show that heparin ran bincl a large amount of cadavwine. HISTAMIXE

BINDIKG

It is known that changers can bind was of interest to t&n:: containing a

RT

0 2

52 56 50

531) Cadaverinr, vt
54 i I

spermine spermine spermidine spermidine cadaverine serotonin serotonin L-adrenaline L-adrenaline Compd. 48-80 550 pg. Compd. 48-N 1000 pg. Compd. 48-80 2000 pg. of anrine

;

43, 47, 47, 49, 53, 54, 54 54, 56, 56, 56, 56, 62. 63

3

~'ROTEISS

carboxylic acid ion exhistamine. Therefore, it determine whether prohigh percentage of glu-

LO7

= 102

156, 159 168. 172 Iv.

= 164

1113i1

.i,M

98, 167 li4. I!)3 Av.

126, 141 161) 201

= 158

Av.

= 157

t’amic and aspartic acids could bind hist’amine. The proteins wzre tested at a concentration of 4 mg./ml. against 2 and 1000 pg. histamine in 19 ml. diffusate. The result’ c> sholvn in Table VI, indicat,e little binding of liistaininc by proteins and support the data of carlic~r workers (51 who also failed t’o clt~monstrntc histamine binding by proteins. HISTAMISE:

BINDISG IIEPSRIK

BY

DESCLFATED

When desulfated heparin, 4 mg./ml., was substituted for hcparin, t’liere was no w-i-

26

KOBAYASHI TABLE HISTAMINE

VI

BINDING

BY PROTENS

Conditions: As described under was 18-20 hr. when 2 pg. histamine 48 hr. when 1000 pg. was used. Per cent of amino acid in protein” Protein

~Fi

Methods. Time was used and

Per cent histamine bound

pg., in 1Y ml. diffusate Asppici ‘Histamine, -__ _ 2

Casein Edestin Zein Gliadin

21.8 19.2 i 31.3 ~ 43.7

10.5 1 10.2 / 1.8 ~ 0.8

5, 4, 2, i 9,

6, 6, 7 Av. Av. 7 2 Av. 9 Av.

1lJoo 6 6 2 9

Ob 06 0” 0”

u From Ref. (8). * Average of 4 values.

dence of histamine binding when only 2 pg. histamine was used in 19 ml. diffusate. The experiment was repeated four t’imes with the same result. The data presented essentially confirm the earlier data of Amann and Werle (3) which demonstrated t’hat heparin dissolved in l$L acetic acid can bind histamine, and that this binc!ing can be inhibit’ed by some polyamines and Compound 48-80. The data presented demonstrated further that this binding of histamine by heparin can occur in distilled water and appears selective in that histamine is bound by heparin in competition with other aminea. The data suggcst that the histamine binding by heparin is due primarily t,o an ion-exchange mechanism as postulated by Amann and Merle. The specificity of t’hc ion exchanger, heparin, is shown by it,s selective binding of liistaminc in tlic presence of equivalent amounts of cadavcrinc, serotonin and Ladrenaline (Table IV). The displacement of liistaminc from heparin by inorganic ions is demonstrated by the action of increasing concentrations of KaCl !Table I). At equal ionic strength, t,lie multivalent ions are more effective than univalent ions in preventing histamine binding, and this would be the expected cast in ion-exchange chromatography. These observations also suggest that others have probably failed to demonstrate

binding because the salts of the buffer solutions used in the experiments prevented binding of histamine by heparin. The data presented in Figs. 1 and 2 indicate t’hat there is a linear relationship bet’ween heparin and the amount of histamine bound. From Fig. 2, it can be seen that 1 mg. heparin bound approximately 200 pg. hist’amine, or 2.0 pg. histamine/i.u. heparin which is coml~arable to that found in rat mast cells ( 1) . Based on a molecular weight of 12,000, 1 mole hcparin will bind 21.5 moles histamine according to the above figurw. Werle and Amann (4) have calculated t’hat 1 mole heparin would bind 22 moles heparin. The chromatographic evidence (Fig. 4) indicated t,hat histamine was bound tightly enough to heparin so that the complex behaves as a single compound. Amam and \\:erle (,3) have demonstrated that a histanlinc-hcparin complex can bc precipitated from alcoholic medium, and also that the complex can be cllromatogral)lled on paper. Sanyal and \;l’est I 7) have also reported the in llitro formation of a histamine-heparin complex. The importnncc of the sulfate molcculc of hcparin in the binding process was shown by the inability of clesulfated heparin to bind histamine. Proteins with a high content, of glutamic and aspartic acids were tested for their abilit’y to bind histamine to dctcrmine whct,hcr an ~~xccss of cnrboxyl groups conferred on proteins the histaminebinding property which the sulfate molecule apparently contributed to heparin. These proteins shoned only a small degree of histaminc binding wlicn a riiinuk conccntration of histamine was in the diffusate, and no binding at a high concentration of histamine. The small binding observed for the proteins at the low histamine concentration was probably tiw to Donnan equilibrium. Howcvcr, for heparin, the large amount of histamine bouncl indicated that the binding ineclianisiii dew not involve the Donnan equilibrium. This is also supported by the observation that lioparin will bind liistamine on a paper strip in the absence of a membrane. It should be noted that the binding capacity of heparin was reduced but not climinateti in 0.01 31 SaCI (Table III) as n-oultl be the case in an ion wchnngctr. The

HISTAMIXE

BINDIKG

binding capacity still remained at a relatively high level of approximately 17 moles histamine/mole heparin. The data of Tables II and III indicate that other factors besides ionic strength arc probably involved in the observed effects of thcsc cations on hist~aruinc binding by heparin. The dat,a presented support t,hc contention that heparin in the granules of the mast cells is probably the histamine-binding agent I,1) and that the mrchanim of this binding is as an ion exchungcr t,3, 4). The clatn further suggest’ that the degree of histaminc binding iuay 1w controlled by the regulation of the internal catiouic environiuent~ within tlrc mast’ rell granules and the aiuount, of liqmrin rontained within these g~~uulw. This postulate is supported by the olwrration that, hcparin n-ill not bind histauiinc in Tyroclc’s solution, and, furthern~or~‘. the liirt~riliiric-llcl~:~l,i~i complex nil1 tlismriatc~ instantly when placed into Tyrotk’s wlutiori. Further lvork is in progrws to study the nature of aiuinc binding by helmin, and the

BY

HEPXRIS

action of various ‘iselectiye” on the bound amincs.

“7

release agents

-1CIiSOWLEDGMESTS The author wishes to acknowledge the tcvhnicnl :&stance of Lorraine E. Leja, Carl 15. Aronron, and I,cSrttn h/l. Shaw. The author is also intlchtetl to Dr. ~hlomo Burstein for many hell)ful sllggestions antI Atid crnluntion of this manuscript.

1. RILET, J. E’., “The Mast Cells.” 1’. and S. Livingstonc, I.ttl., Etlinhurgh, 1959. 2. hI.\(,IxToslI, F. C., i)~ “Cikxl Symposium on Histammc~.” Lit tic, Brown and Co., Boston, 1956.

11. n-.. SMILET, R. I*., AND IiEsser)r, ,J., d. N/rd. C’lie,,/. 206, 461 (1954). 6. Ii.\~~rr, h:., .ISD Da\-IS;, J., I-‘IYIc,. Sot. Erptl. Bid. .llcd. 84, 218 (1953). 7. S.\\I-.a~,, It. Ii., .IAII) Wl:sr, G. B., Sollcye 178, 1293 ( 1956). 8. &x\rIDT, C. I,. A1., “The Chemistry of the Amino .\citls an(l Proteins.” Charles C Thomas, ,s;lll’illefidd, lW5. 5. S(‘II.\~X,