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The antagonistic activities of conalbumin and 8-hydroxyquinoline (oxine)
The Antagonistic Activities of Conalbumin and 8-Hydroxyquinoline (Oxine) Robert E. Feeney From the Western Regional Research Laboratory,1 Albany, California Received March 20, 1951 INTRODUCTION
In a study of the mechanism of action of conalbumin, an egg white protein (2), it has been found that the inhibition of growth of several gram-positive bacteria caused by either conalbumin or 8-hydroxyquinoline (oxine) does not occur when these two substances are added together to the test culture media. These apparently antagonistic activities are described and discussed. Both conalbumin and oxine form complexes with metal ions, and their antibacterial activities are caused by the formations of their complexes. Conalbumin forms a stable complex with iron, and the inhibition of bacterial growth caused by its addition to media is specifically prevented by the addition of iron salts (6,12,13). In contrast, various metal ions complex with oxine, and, as reported recently by Rubbo, Albert, and Gibson (10), several metal ions nonspecifically prevent the inhibition of certain bacteria by oxine. However, a specificity occurs with gram-positive bacteria and Neisseria. Cobalt is unique among cations in antagonizing the action of oxine against these organisms. This same report also makes the important observation that the inhibitory activity of oxine for these organisms does not occur in media from which the ~race elements have been removed by extractions with chloroform solutions of oxine. The antibacterial activity of oxine is thus not simply caused by a deficiency of.cobalt but is dependent on the presence of other trace elements (10). The results of the present study indicate that the antibacterial activity of conalbumin, as well as that of oxine, may be dependent on the i Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 196
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p r e s e n c e of o t h e r t r a c e e l e m e n t s . T h e a b s e n c e of i n h i b i t i o n o b s e r v e d i n c e r t a i n m i x t u r e s of c o n a l b u m i n a n d o x i n e is i n t e r p r e t e d o n t h i s basis.
METHODS The general bacteriological procedures were as previously described (6). Tests were conducted in 5-ml. volumes of medium. The base medium as-finally diluted in the test cultures had the following composition: 0.40% Bacto-peptone, 0.12% Bactomeat extract, 0.50% disodium phosphate, and 0.03% citric acid. Protein solutions were sterilized by filtration and added to the medium after sterilization. The phosphate was also sterilized separately and added to the medium after sterilization, to reduce hazards of precipitation. The pH of the medium after sterilization and addition of phosphate and protein solutions was 7.5-7.6. All salts and reagents used were of reagent grade. Redistilled water was used throughout. Trace elements were added as chlorides or sulfates. The conalbumin preparations were similar to those previously employed (6) (10 rag. bound approx. 11.0 #g. of iron). In calculating the molar concentrations employed, the older value for the molecular weight, 87,000 (3), was employed rather than the newer value, 77,000 (14), for crystalline material. The ovalbumin preparations were similar to those recently employed for other studies in this laboratory (7). The cultures of gram-positive bacteria were as follows: Micrococcus pyogenes vat. albus (6), Bacillus subtilis (4), and Micrococcus lysodeikticus (1). Unless otherwise indicated, results reported were obtained with M. pyogenes var. albus as the test organism. The gram-negative bacteria were laboratory-identified strains of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Proteus vulgaris, and an unidentified organism isolated from spoiled eggs. Inocula were grown at 37~ for 18-24 hr. on nutrient broth. They were diluted 10,000-fold in saline, and 0.1 ml. of the saline dilution was employed per 5 ml. of test medium. Ps. fluorescens was grown at 250~ Growth of M. pyogenes var. albus was usually determined on triplicate tubes by turbidity determinations on a Klett-Summerson photoelectric colorimeter. Figures given are averages of the readings of the three tubes. The tubes were shaken manually and read at various periods during incubation. Relative growths of the other organisms and occasionally of M. pyogenes var. albus were estimated visually. Iron was determined as previously described (6). Copper and cobalt were determined by the methods of McFarlane (8) and McNaught (9). The experiments in which the conalbumin and bacteria were separated from one another by a dialysis membrane were performed in a series of sterile tubes referred to as dialysis tubes. These dialysis tubes consisted essentially of an inner tube, With a bag of dialysis tubing attached, inserted into an Outer, wide-diameter test tube. The cellophane tubing (approx. 1.5 cm. in diameter) was tied at one end to form a bag. It was then pulled over 6-7 cm. of a 10-cm'. section of Pyrex glass tubing (app. 1.5 cm. in diameter) so that the volume in the bag was 6-7 ml. The bag and lower part of the glass tube were next soaked for 48-72 hr. in 0.1 M citric acid and for 24 hr. in two changes of redistilled water. A band (2-3 cm. wide) of relatively lint-free gauze was wrapped around the glass tube at a point beginning approximately 2 cm. below the upper end of the tube. The band served asthe plug for inserting and holding
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ROBERT E. FEENEY
this tube in the outer test tube. A cotton plug was then placed in the mouth of the tube, and the tube was inserted into the outer test tube. This test tube was 3 cm. in inside diameter and 10-11 cm. in height and contained 20 ml. Of water prior to inserting the tube and bag. The height of the bag was adjusted by properly positioning the inner tube so as to suspend 9 the bag. This assembled dialysis tube was sterilized b y autoclaving. Solutions of test reagents and 10-times-strength medium were sterilized separately. The test reagents, 2.5 ml. of the medium, and sufficient water to give 5 ml. of total volume were then aseptically added to the inner tube. The dialysis tube was then incubated for 72 hr. at 37~ for equilibration. Finally the tube was inoculated inside or outside the bag as desired.
RESULTS Growth Inhibitions by Conalbumin and Oxine I n t h e s e s t u d i e s c o m p l e t e i n h i b i t i o n of g r o w t h w a s O b t a i n e d a t c o n c e n t r a t i o n s of c o n a l b u m i n of > 4.6 ~ M . T h e m i n i m u m c o n c e n t r a t i o n s TABLE I
Interrelationships of Conalbumin, Oxine, Iron, Cobalt, and Copper Additions to tubes Tube set
T u r b i d i t i e s a for m e d i a a n d h o u r s of i n c u b a t i o n i n d i c a t e d b e l o w Base b
Cons bumi
#mol~.
0 1.4 2.3 4.6 6.9 6.9 0 2.3 2.3
0 0 0 0 0 18(Fe) 16(Cu) 16(Cu) 17(Co)
B a s e + oxine + C o c
I 16
~mole~
B a s e + oxine ~
Other
~0 31 13 0 0i 36 28 8 0
19 --
22 --
39
48
45 43 23 0 0 47 37 15 4
59 80 93 53 80 94 39 58 83 0 0 0' 0 0 0' 59 79 89 49 72 88 26 52 72 7 19 20
16 I 19
22
39
39
48
48
0
0 0 0 0 0 a 29 0 0 0 0 0 a 30 0 11 26 60 83 11 6 15 31 63 80 8 20 52 58 7 0 0 0 0 0 0 a 3! 0 0 0 0 Oa[ 0 0 0 0 Oa!
40 50 64 66 40 51 63 64 19 3(~ 50 59 0 0 0 0a 0 0 0 0~ 40 50 58 57 13 21 40 37 4 7 17 18
Turbidities were measured on a Klett-Summerson photoelectric colorimeter. Figures are averages of triplicate tubes. b Composition of medium described in text. The iron, copper, and cobalt contents of this medium were 0.21 p.p.m., approx. 0.017 p.p.m., and approx. 0.01 p.p.m., respectively, as determined b y chemical analyses on an ash of the medium constituents. These concentrations were: 3.8, 0.27, and 0.17 ~moles/liter. Inoculum was 0.1 ml. of a 1 : 10,000 dilution of a 24-hr. broth culture. c Oxine and cobalt concentrations were each 1.0 p.p.m, or respectively 6.9 ~M a n d 17 uM. a No g r o w t h occurred in these tubes after 120 hr. of incubation.
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at which a decrease in growth rate was observed were 1.7-2.0 ~M. This value is chemically equivalent to an iron concentration of 3.4-4.0 ~M [-1 mole of eonalbumin complexes with 2 moles of iron (6)-]. The value agreed closely with the analytical value for iron in the medium of 3.6 ~M (see Table I). The minimum concentration of oxine which inhibited growth was >2.8 t~M and <4.2 t~M. This concentration was approximately 20 times the theoretical concentration necessary to complex with the cobalt found by chemical analysis and almost twice that required for the total iron, copper, and cobalt (Table I). A combining ratio of 1 mole of oxine to 2 moles of metal ion was employed for these estimatibns (10). A value approximating this ratio was obtained by determining the conCentration of cobalt required to prevent inhibition of growth caused by excess oxine. In such an experiment a cobalt concentration of approximately 6.8 ~M was required for a total oxine concentration of 6.9 ~M or >2.7 and <4.1 ~M of excess oxine. These figures give a calculated combining ratio of > 1.6 and <2.4 moles of cobalt/mole of oxine. None of the following elements prevented inhibition by 6.9 ~M oxine when tested individually at the indicated ~M concentrations: Cu, 16; Mn, 18; Fe, 18; Zn, 15; Mo, 10; and Mg, 41.
Interrelationships of Conalbumin, Oxine, Iron, and Cobalt When conalbumin, oxine, iron, and cobalt were tested as various combinations and at varying concentrations, well-defined and" quantitative relationships were found. In essence, conalbumin and oxine had little or no inhibitory activity when tested together under certain conditions wherein both were inhibitory when tested singly. Thus, this apparent mutual counteraction of inhibitory activities only occurred at concentrations of both conalbumin and oxine which were inhibitory when added alone. The further addition of either or both of the specific metal ions (iron and/or cobalt), in concentrations less than those required to prevent the inhibition normally given by the particular concentration of complexing agent employed, had no effect. However, the addition of either ion in a concentration sufficient to prevent inhibition by its complexing agent also prevented the counteraction of inhibitory activity. Finally, the addition of both metal ions in concentrations sufficient to prevent inhibitions by their complexing agents again allowed for growth.
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E. FEENEY
The results of one of more than 20 experiments on this subject are presented in Table I. From inspection of the data it can be seen that only slight inhibition (as evidenced only by an increase in the lag period) was obtained with an excess of both conalbumin and oxine (tube sets 3, 4, and 5, Table I). With further addition of either iron or cobalt, inhibition was again obtained; but with further addition of both iron and cobalt, growth occurred (tube sets 4, 5, and 6). These data also show how the counteraction of oxine inhibition was not obtained with an amount of conalbumin which would not at least partially inhibit growth when added alone (tube set 2). Similar quantitative relationships were always found in other experiments. Also, similar quantitative relationships for oxine and cobalt were found. The minimum concentration of oxine necessary to allow for growth when tested with 6.9 tLM conalbumin was the same as that necessary to cause inhibition, >2.8 t~M. The addition of 3.4 pM cobalt or 3.6 ~M iron did not influence the growth given by a mixture of 6.9 t~M conalbumin and 6.9 pM oxine. The above-described, apparently mutually antagonistic activities of conalbumin and oxine did not occur at concentrations 10-20 times greater than the minimum concentrations necessary to give inhibition when tested singly. The concentrations tested were 23 t~M and 46 t~M for conalbumin and 28 ~M for oxine. Prevention of inhibitory activities, however, was given by equivalent concentrations of iron or copper when the complexing agents were tested singly. Studies on the effects of high concentrations of iron and cobalt were attempted but were discontinued because of the formation of visible precipitates. Concentrations of iron up to 72 ~M (approx. 20 times concentration of iron in medium) were tolerated, but a cobalt concentration of 68 ~M (approx. 400 times concentration in medium) was partially inhibitory. A very slight precipitation was caused by this concentration of cobalt. The inhibition was largely prevented by 14 or 28 ~M oxine but not by 72 ~M iron. Lower concentrations of cobalt increased the inhibition given by borderline concentrations of conalbumin (tube set 9, Table I).
Effects of Other Iron-Binding Agents and Proteins Two other substances capable of complexing with iron were found to substitute at least partially for conalbumin in giving the mutual
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201
counteraction of inhibitions with oxine. The one, hydroxylamido ovomucoid, an iron-binding derivative of the egg white protein ovomucoid (5), inhibited growth at concentrations > 60 ~g./ml. ; the inhibition was prevented by the addition of iron but not by other metal ions tested. Inhibition was also prevented by the addition of 6.9 ~M oxine; in all other respects studied, the synthetic complexing agent behaved like conalbumin in mixtures with oxine. The other complexing agent, o-phenanthroline, inhibited growth at concentrations > 11 ~M. When it was tested at 22 ~M with 6.9 ~M oxine, a counteraction of inhibitory activities occurred, but only slow and slight growth was obtained. However, this result was considered confirmatory because inhibition by o-phenanthroline was also only poorly prevented by the addition of iron. To test the possibility that a protein per se, or various of its reactive groupings, might nonspecifically have effects similar to those of conalbumin, crystalline ovalbumin was tested. At concentrations of 0.45, 4.5, and 90 t~M, ovalbumin had no effect on the inhibition caused by 6.9 ~M oxine.
Interrelationships When the Conalbumin and Oxine Were Separated by a Dialysis Membrane These experiments were conducted with the dialysis tubes described under the Methods section. In a Preliminary series of eight experiments, these tubes were employed to determine whether or not conalbumin would inhibit growth when the conalbumin was added inside the bag and the medium was inoculated outside the bag. This proved to be the case, ~ and the addition of iron prevented this inhibition. In a further series of four experiments on the interrelationships of oxine and conalbumin, it was found that the counteraction of inhibition in conalbuminoxine mixtures also occurred when the bacteria were separated from the conalbumin (see Table II).
Effects of Other Metal Ions; Media and Cultural Conditions Several factors were found to influence to varying degrees the interrelationships between conalbumin and oxine and the inhibitions given 2 In several earlier experiments, more rapid growth occurred when the organisms were separated from the conalbumin. However, these experiments were discounted
because they were performedwith dialysistubes and bags which had not been soaked in citrate solution.
202
R O B E R T E. F E E N E Y
TABLE II
Expt.
Interrelationships with Organism Separated from Conalbumin by a Dialysis Membrane Relativegrowthr Additions~to medium inside bag Inoculated b after hours indicated 16 24 41 65
A
None Conalbumin Oxine Conalbumin -{- oxine Conalbumin Conalbumin + oxine
Outside bag Do. Do. Do. Inside bag Do.
B
N one Conalbumin Conalbumin -{- oxine
Outside bag Do. Do.
2 0 0 0 0 0
4 0 0 tr 0 1
4 0 0 2 0 2
-0 0 3 tr 3
17
22
41
66
3 0 1
4 0 2
0 4
tr 4
The medium and PreParation of dialysis tubes are described in text. The pH of the medium in these experiments was 8.0. The total volume inside and outside the dialysis bag was 25 ml. The concentrations of conalbumin and oxine added were respectively 4.6 ~M and 6.9 ~M as calculated on total volume of medium. b Outside bag and inside bag refer to site of inoculation. Inocula were 0.1 ml. of a 1 : 10,000 dilution of a 24-hr. broth culture in distilled water. c Growth was estimated visually and reported as tr = trace, and 1 to 4 = gradations from slight to maximum growth. In each case the growth reported refers to media inside or outside of bag corresponding to site of inoculation. The other section always remained sterile. b y t h e s e m a t e r i a l s . E x t e n s i v e effects w e r e o b t a i n e d b y a d d i n g o t h e r m e t a l ions in t h e p r e s e n c e of e i t h e r or b o t h of t h e m e t a l c o m p l e x i n g a g e n t s . C o p p e r i n c r e a s e d i n h i b i t i o n b y c o n a l b u m i n , oxine, or a m i x t u r e of t h e t w o ( t u b e sets 7 a n d 8, T a b l e I ; T a b l e I I I ) . I n o t h e r e x p e r i m e n t s complete inhibition was not obtained with copper, conalbumin, and oxine as in t u b e set 8, T a b l e I . M a n g a n e s e p a r t i a l l y p r e v e n t e d t h e effect of c o p p e r in o x i n e - c o b a l t m i x t u r e s (.Table I I I ) . M o l y b d e n u m a n d m a g n e s i u m h a d l i t t l e effect, while zinc w a s s t r o n g l y i n h i b i t o r y e v e n in t h e a b s e n c e of t h e c o m p l e x i n g a g e n t s . That the conalbumin-oxine system was delicately balanced was s h o w n b y e x p e r i m e n t s on t h e effect of c h a n g i n g t h e size of t h e i n o c u l u m . I n c r e a s i n g t h e size of t h e i n o c u l u m 100-fold i n c r e a s e d t h e c o n c e n t r a t i o n of c o n a l b u m i n r e q u i r e d t o c o m p l e t e l y i n h i b i t g r o w t h f r o m < 4.6 ~ M t o > 6.9 u M b u t , p o s s i b l y a s a d i r e c t c o n s e q u e n c e of t h i s d e c r e a s e in sensit i v i t y of t h e o r g a n i s m t o c o n a l b u m i n , d e c r e a s e d t h e r a t e of g r o w t h in
CONALBUMIN AND OXINE
203
c o n a l b u m i n - o x i n e m i x t u r e s . C h a n g i n g t h e s o u r c e of t h e a n i m a l p r o d u c t c o n s t i t u e n t s of t h e m e d i u m or a d d i n g t h e p h o s p h a t e p r i o r to s t e r i l i z a t i o n also c h a n g e d t h e g r o w t h in c o n a l b u m i n - o x i n e m i x t u r e s .
Comparative Studies with Other Bacteria Several organisms which were inhibited by c o n a l b u m i n by varying d eg r ees w e r e s e l e c t e d for t h e s e studies. All of t h e s e o r g a n i s m s w e r e TABLE III
Effects of Zinc and Manganese on Inhibitions by Conalbumin and Oxine Expt.
A
B
C
Tube set 1 2 3 4 5
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5
Additionsa to medium None Oxine Oxine, Co Oxine, Co, Cu Oxine, Co, Cu, Mn
None Conalbumin Conalbumin, Co Conalbumin, Cu Conalbumin, Co, Conalbumin, Zn, Conalbumin, Co, Conalbumin, Co, Conalbumin, Co, Co, Cu Zn, Mn Co, Cu, Zn, Mn
Cu Mn Cu, Zn, Mn Cu, Zn Cu, Mn
None Conalbumin Conalbumin, Zn Conalbumin, Zn, Co Conalbumin, Zn, Co, Cu, Mn
Turbiditiesbafter hours indicated below 16 24 40 66 35. 0 30 0 19
63 0 47 5 31
76 0 57 22 41
97 0 70 29 50
18
25
42
66
38 16 1 6 0 0 0 0 0 31 2 30
62 36 18 25 0 0 0 0 0 55 31 52
78 54 39 38 3 0 0 0 9 74 68 63
99 79 59 56 47 0 0 0 35 87 78 74
17
25
41
116
40 23 0 0 0
62 45 0 0 0
76 55 0 0 0
--0 0 0
Concentrations of materials added expressed as #moles/liter were: oxine, 6.9; conalbumin (Expt. B), 2.3; conalbumin (Expt. C), 2.0; Co, 17; Cu, 16; Mn, 18; and Zn, 15. b Composition of medium described in text and Table I. Turbidities were measured on a Klett-Summerson photoelectric colorimeter. Figures are averages of triplicate tubes.
204
ROBERT
E.
FEENEY
i n h i b i t e d t o a t l e a s t s o m e e x t e n t on t h i s m e d i u m in t h e p r e s e n c e of a large excess of c o n a l b u m i n ( > 23 ~M). M . lysodeikticus w a s c o m p l e t e l y i n h i b i t e d b y 2.0 ~ M c o n c e n t r a t i o n of c o n a l b u m i n , w h i c h w a s o n l y s l i g h t l y in s t o i c h i o m e t r i c excess of t h e i r o n in t h e m e d i u m a n d signific a n t l y less t h a n t h e c o n c e n t r a t i o n ( a p p r o x . 3.5 ~ M ) r e q u i r e d t o i n h i b i t c o m p l e t e l y M . pyogenes v a r . albus u n d e r t h e s e c o n d i t i o n s . B. subtilis, h o w e v e r , while its g r o w t h r a t e was p r o g r e s s i v e l y r e t a r d e d as t h e c o n c e n t r a t i o n of c o n a l b u m i n w a s i n c r e a s e d a b o v e 2.0 ~ M , r e q u i r e d r e l a t i v e l y h i g h c o n c e n t r a t i o n s ( > 23 ~ M ) for c o m p l e t e i n h i b i t i o n . B o t h of TABLE IV
Interrelationships of Conalbumin, Oxine, Iron, and Cobaltfor Bacillus subtilis and Micrococcus lysodeikticus Tube set
1 2 3 4 5 6 7 8
Additions to medium a
None Conalbumin (1.6) Conalbumin (4.6) Conalbumin (4.6), Fe (21) Oxine (6.9) Oxine (6.9), Co (17) Conalbumin (1.6), oxine (7) Conalbumin (1.6), oxine (6.9), Fe (7)
Growth b of organisms after hours indicated B. subtilis M . lysodeikticus
i6
66
2 2 tr 2 0 2 0 0
4 4 4 4 0 4 0 0
140 4 4 4 4 0 4 1c 0
16 66 1 tr 0 tr 0 tr tr 0
4 3 0 4 0 4 3 0
!40 4 3 0 4 0 4 4 0
Composition of medium described in text and in footnotes of Table I. Figures in parentheses signify concentrations in ~moles/liter of materials added. b Inocula were 0.1 ml. of a saline suspension of a 1 : 10,000 dilution of a 24-hr. broth culture. Growth was estimated visually on duplicate tubes and reported as: tr = trace, and 1 to 4 = gradations from slight growth to maximum. c Good growth in oxine-conalbumin mixtures was obtained in other experiments with B. subtilis where the higher level of conalbumin (4.6 ~M) was employed. t h e s e o r g a n i s m s were i n h i b i t e d c o m p l e t e l y b y 6.9 p M oxine ( T a b l e I V ) a n d s h o w e d t h e m u t u a l c o u n t e r a c t i o n of i n h i b i t o r y a c t i v i t i e s in c o n a l b u m i n - o x i n e m i x t u r e s , b u t in b o t h cases t h e c o n c e n t r a t i o n s of c o n a l b u m i n r e q u i r e d for t h i s c o u n t e r a c t i o n were d i r e c t l y r e l a t e d t o t h e c o n c e n t r a t i o n s r e q u i r e d for i n h i b i t i o n . T h u s , g o o d g r o w t h of M . lysodeikticus o c c u r r e d in c o n a l b u m i n - o x i n e m i x t u r e s a t 1.6 ~ M c o n a l b u m i n ; t h i s w a s l o w e r t h a n t h e c o n c e n t r a t i o n o p t i m u m for M . pyogenes v a r . albus(see T a b l e I). F o u r o t h e r b a c t e r i a s e l e c t e d were g r a m - n e g a t i v e r o d s w h i c h were o n l y s l i g h t l y i n h i b i t e d b y c o n a l b u m i n . A s r e p o r t e d b y R u b b o et al. ( t 0 ) ,
CONALBUMIN AND OXINE
205
a concentration of oxine as high as 27 t~M did not inhibit the gramnegative rods. This concentration of oxine also did not appear to influence any inhibition of these organisms by conalbumin.
Complexing of Copper, Cobalt and Zinc with Conalbumin The formation of complexes of copper, cobalt, and zinc with conalbumin was of obvious interest in these studies. It has been previously reported that copper forms an easily dissociated yellow complex and cobalt does not form a colored complex (6). Results of this same study also indicated that the weak linkage with copper involved the same reactive groups and properties of the protein as the strong linkage with iron involved. In fact, the yellow color of solutions of the copper complex was quickly displaced by the salmon-pink color of the iron complex upon the addition of iron. On the basis of this information, the effects of cobalt and zinc on the development of the copper color were studied in an attempt to determine the combining capacities of conalbumin for cobalt and zinc relative to its weak capacity for copper. Neither cobalt nor zinc gave any color when tested at concentrations of 80-100 ~M with 11.5 t~M conalbumin in phosphate-bicarbonate-citrate buffer (6) at pH 7.6. Similar concentrations in phosphate-bicarbonate buffer at pH 7.6 also did not influence the development of the yellow color upon the addition of 25 ~M copper. The complexing of conalbumin with cobalt or zinc, therefore, appeared to be, at the most, extremely weak. DISCUSSION
The studies of Rubbo et al. (10) on the action of oxine clearly show an important role for iron and copper in the inhibitory action of oxine with gram-positive bacteria. Their results demonstrate the absence of toxicity of oxine on oxine-extracted media, the presence of toxicity of oxine on oxine-extracted media when iron and copper are added, and the absence of oxine toxicity when the oxidation-reduction (redox) potential of the medium is lowered by the addition of ascorbic acid. They support the hypothesis that the toxic effects of oxine are due to the action of lipide-soluble, toxic, oxine complexes of iron or copper per se and not to the free metal ions, The beneficial effects of cobalt on growth under their conditions are attributed to its prevention of the actions of these oxine complexes of iron or copper and not to the prevention of their formation.
206
ROBERT
E. F E E N E Y
The results of the present investigation can be interpreted as confirming the dependence of oxine activity on the presence of other trace elements. They can also be interpreted as showing tha t the antibacterial activity of conalbumin is likewise dependent on other trace elements. Such interpretations require the assumption that the absence of inhibitions in mixtures of these materials is caused by their metal-binding properties. The observed quantitative interrelationships support such an assumption rather than some less evident possibility such as chemical interreaction between the conalbumin and oxine. The results are not easily explained, however, on the basis of the formation of toxic complexes as proposed for oxine (10). Conalbumin binds copper so loosely that the complex is dissociated with citrate (6). The toxic complex of oxine with copper advanced by Rubbo et al. should therefore exist in the presence or absence of conalbumin, and conalbumin should not counteract oxine inhibition by binding the copper. Although the addition of copper to conalbumin-oxine mixtures was strongly inhibitory, there should have been ampl e copper in the medium to form a toxic complex with oxine. :While there is no a priori reason why two different metal ion complexing agents should inhibit bacterial growth by similar mechanisms, a comparison of their possible mechanisms of action should aid in understanding these mechanisms. The results with conalbumin were such that its inhibitory activity could not be interpreted on the basis of the formation of a toxic complex. Conalbumin apparently does not form stable complexes with either cobalt or copper and its complexes are not lipide-soluble. Finally, the demonstration that conalbumin inhibits bacterial growth when the protein and bacteria are physically separated by a cellophane membrane eliminates any possibility of a direct action of a complex on the organism. These considerations and results not only necessitate a different hypothesis for conalbumin activity but also strongly suggest that a different hypothesis is necessary for oxine activity. The results of this investigation might be interpreted as further examples of the deleterious effects resulting from imbalances in the concentrations of trace elements. The inhibition of the growth of M. pyogenes var. albus by conalbumin on the medium studied would thus be caused by the binding of iron and the resulting relatively low concentrations of iron and relatively high concentration of cobalt available to the organism. Likewise, the inhibition by oxine would be caused by relatively low concentrations of cobalt and relatively high concentra-
CONALBUMIN
AND OXINE
207
tions of iron. Finally, the absence of inhibitory activities when conalbumin and oxine are added together Would result from the simultaneous lowering of the active concentrations of both iron and cobalt and the consequent absence of an imbalance. The fact that growth does not occur at high levels of conalbumin or oxine in conalbumin-oxine mixtures indicates that a small, but criticai, amount of the active element is necessary and thus favors an imbalance interpretation. Obviously, such a simple interpretation as an imbalance does not explain why the imbalance is toxic. Nevertheless, despite these considerations, the toxic effects of copper and zinc observed in this study are difficult to explain unless one hypothesizes other imbalances involving these ions. The addition of copper or zinc, and possibly other metal ions, would thus cause new imbalances. The studies of Schade (11) on the influence of media constituents on cobalt toxicities further illustrate the manifold factors to be considered in such relationships. In the present study, sulfhydryl groups of media constituents were not considered to play a primary role in the conalbumin-0xine interrelationships. Conalbumin apparently has no sulfhydryl groups, and ovalbumin, at 90 ~M, a concentration sufficient to give approximately 300 ~M sulfhydryl (7), did not affect inhibition by oxine or growth in the absence of oxine. The possibility that chemical interactions involving conalbumin and oxine are responsible for these antagonistic effects has not been eliminated. Unpublished studies of the author in cooperation with S. S. Elberg and W. S. Waring of the University of California indicate some very slow interactions under specific conditions, as previously reported for an excess of o-phenanthroline and the conalbumin-iron complex (6). However, it appears difficult to interpret these effects as mutual inactivation. There are at least two advantages of such a specific metal-binder as conalbumin for studies of this nature. In order to similarly change the concentrations of metal ions by the direct addition of salts to media, it would be necessary to add such high levels that non-specific effects such as precipitations might occur. Another advantage is that the protein or its complexes do not pass through dialyzing membranes; thus intimate contact of complexing agent and organisms is eliminated. The interpretations of these results are important in considering the antibacterial properties of egg white and the role of conalbumin therein
208
ROBERT E. FEENEY
(12, 13). They indicate that variations in contents of trace metal ions other than iron might strongly influence the antibacterial activity. ACKNOWLEDGMENTS The author gratefully acknowledges the assistance of David A. Nagy throughout this investigation, the performance of the copper and cobalt analyses by E. F. Potter, the preparation of the hydroxylamido ovomucoid by Heinz Fraenkel-Conrat, and the performance of several analyses bY E. D. Ducay. SUMMARY
The growth of three gram-positive organisms was inhibited by addition of either conalbumin or oxine (8-hydroxyquinoline). This inhibition did not occur with further addition of iron in the case of conalbumin, and of cobalt in the case of oxine, or with the addition of both conalbumin and oxine together. When conalbumin and oxine were added together, inhibition was again obtained by adding either cobalt or iron but was not obtained by adding both cobalt and iron. The significance of these findings is discussed. I~EFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. ]0. 11. 12. 13. 14.
ALDERTON, G., AND FEVOLD, H. L., J. Biol. Chem. 164, 1 (1946). ALDERTON,G., WARD, W. H., AND FEVOLD, H. L., Arch. Biochem. 11, 9 (1946). BAIN, 5. A., AND DEUTSCH, H. F., Or. Biol. Chem. 172, 547 (1948). FEENEY, R. E., AND GARIBALDI,J. A., Arch. Biochem. 17, 447 (1948). FRAENKEL-CONRAT,H., Arch. Biochem. 28, 452 (1950). FRAENKEL-CONRAT,H., AND FEENEY, R. E., Arch. Biochem. 29, 101 (1950). MACDONNELL,L. R., SILVA,R. B., AND FEEI~Eu R. E., Arch. Biochem. Biophys. 32, 288 (1951). MCFARLANE,W. D., Biochem. J. 26, 1022 (1932). McNAUGHT, K. J., Analyst 67, 97 (1942). RUBBO, S. D., ALBERT, A., AND GIBSON, M. I., Brit. J. Exptl. Path. 31, 425 (1950). SCHADE,A. L., J. Bact. 58, 811 (1949). SCHADE, A. L., AND CAROLINE, L., Science 100, 12 (1944). SCHADE,A. L., AND CAROLINE, L., Science 104, 340 (1946). WARNER, R. C., AND WEBER, I., Am. Chem. Soe. Abstracts of Papers, p. 24C. l l 9 t h meeting, 1951.
In a study of the mechanism of action of conalbumin, an egg white protein (2), it has been found that the inhibition of growth of several gram-positive bacteria caused by either conalbumin or 8-hydroxyquinoline (oxine) does not occur when these two substances are added together to the test culture media. These apparently antagonistic activities are described and discussed. Both conalbumin and oxine form complexes with metal ions, and their antibacterial activities are caused by the formations of their complexes. Conalbumin forms a stable complex with iron, and the inhibition of bacterial growth caused by its addition to media is specifically prevented by the addition of iron salts (6,12,13). In contrast, various metal ions complex with oxine, and, as reported recently by Rubbo, Albert, and Gibson (10), several metal ions nonspecifically prevent the inhibition of certain bacteria by oxine. However, a specificity occurs with gram-positive bacteria and Neisseria. Cobalt is unique among cations in antagonizing the action of oxine against these organisms. This same report also makes the important observation that the inhibitory activity of oxine for these organisms does not occur in media from which the ~race elements have been removed by extractions with chloroform solutions of oxine. The antibacterial activity of oxine is thus not simply caused by a deficiency of.cobalt but is dependent on the presence of other trace elements (10). The results of the present study indicate that the antibacterial activity of conalbumin, as well as that of oxine, may be dependent on the i Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 196
CONALBUMIN AND OXINE
197
p r e s e n c e of o t h e r t r a c e e l e m e n t s . T h e a b s e n c e of i n h i b i t i o n o b s e r v e d i n c e r t a i n m i x t u r e s of c o n a l b u m i n a n d o x i n e is i n t e r p r e t e d o n t h i s basis.
METHODS The general bacteriological procedures were as previously described (6). Tests were conducted in 5-ml. volumes of medium. The base medium as-finally diluted in the test cultures had the following composition: 0.40% Bacto-peptone, 0.12% Bactomeat extract, 0.50% disodium phosphate, and 0.03% citric acid. Protein solutions were sterilized by filtration and added to the medium after sterilization. The phosphate was also sterilized separately and added to the medium after sterilization, to reduce hazards of precipitation. The pH of the medium after sterilization and addition of phosphate and protein solutions was 7.5-7.6. All salts and reagents used were of reagent grade. Redistilled water was used throughout. Trace elements were added as chlorides or sulfates. The conalbumin preparations were similar to those previously employed (6) (10 rag. bound approx. 11.0 #g. of iron). In calculating the molar concentrations employed, the older value for the molecular weight, 87,000 (3), was employed rather than the newer value, 77,000 (14), for crystalline material. The ovalbumin preparations were similar to those recently employed for other studies in this laboratory (7). The cultures of gram-positive bacteria were as follows: Micrococcus pyogenes vat. albus (6), Bacillus subtilis (4), and Micrococcus lysodeikticus (1). Unless otherwise indicated, results reported were obtained with M. pyogenes var. albus as the test organism. The gram-negative bacteria were laboratory-identified strains of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Proteus vulgaris, and an unidentified organism isolated from spoiled eggs. Inocula were grown at 37~ for 18-24 hr. on nutrient broth. They were diluted 10,000-fold in saline, and 0.1 ml. of the saline dilution was employed per 5 ml. of test medium. Ps. fluorescens was grown at 250~ Growth of M. pyogenes var. albus was usually determined on triplicate tubes by turbidity determinations on a Klett-Summerson photoelectric colorimeter. Figures given are averages of the readings of the three tubes. The tubes were shaken manually and read at various periods during incubation. Relative growths of the other organisms and occasionally of M. pyogenes var. albus were estimated visually. Iron was determined as previously described (6). Copper and cobalt were determined by the methods of McFarlane (8) and McNaught (9). The experiments in which the conalbumin and bacteria were separated from one another by a dialysis membrane were performed in a series of sterile tubes referred to as dialysis tubes. These dialysis tubes consisted essentially of an inner tube, With a bag of dialysis tubing attached, inserted into an Outer, wide-diameter test tube. The cellophane tubing (approx. 1.5 cm. in diameter) was tied at one end to form a bag. It was then pulled over 6-7 cm. of a 10-cm'. section of Pyrex glass tubing (app. 1.5 cm. in diameter) so that the volume in the bag was 6-7 ml. The bag and lower part of the glass tube were next soaked for 48-72 hr. in 0.1 M citric acid and for 24 hr. in two changes of redistilled water. A band (2-3 cm. wide) of relatively lint-free gauze was wrapped around the glass tube at a point beginning approximately 2 cm. below the upper end of the tube. The band served asthe plug for inserting and holding
198
ROBERT E. FEENEY
this tube in the outer test tube. A cotton plug was then placed in the mouth of the tube, and the tube was inserted into the outer test tube. This test tube was 3 cm. in inside diameter and 10-11 cm. in height and contained 20 ml. Of water prior to inserting the tube and bag. The height of the bag was adjusted by properly positioning the inner tube so as to suspend 9 the bag. This assembled dialysis tube was sterilized b y autoclaving. Solutions of test reagents and 10-times-strength medium were sterilized separately. The test reagents, 2.5 ml. of the medium, and sufficient water to give 5 ml. of total volume were then aseptically added to the inner tube. The dialysis tube was then incubated for 72 hr. at 37~ for equilibration. Finally the tube was inoculated inside or outside the bag as desired.
RESULTS Growth Inhibitions by Conalbumin and Oxine I n t h e s e s t u d i e s c o m p l e t e i n h i b i t i o n of g r o w t h w a s O b t a i n e d a t c o n c e n t r a t i o n s of c o n a l b u m i n of > 4.6 ~ M . T h e m i n i m u m c o n c e n t r a t i o n s TABLE I
Interrelationships of Conalbumin, Oxine, Iron, Cobalt, and Copper Additions to tubes Tube set
T u r b i d i t i e s a for m e d i a a n d h o u r s of i n c u b a t i o n i n d i c a t e d b e l o w Base b
Cons bumi
#mol~.
0 1.4 2.3 4.6 6.9 6.9 0 2.3 2.3
0 0 0 0 0 18(Fe) 16(Cu) 16(Cu) 17(Co)
B a s e + oxine + C o c
I 16
~mole~
B a s e + oxine ~
Other
~0 31 13 0 0i 36 28 8 0
19 --
22 --
39
48
45 43 23 0 0 47 37 15 4
59 80 93 53 80 94 39 58 83 0 0 0' 0 0 0' 59 79 89 49 72 88 26 52 72 7 19 20
16 I 19
22
39
39
48
48
0
0 0 0 0 0 a 29 0 0 0 0 0 a 30 0 11 26 60 83 11 6 15 31 63 80 8 20 52 58 7 0 0 0 0 0 0 a 3! 0 0 0 0 Oa[ 0 0 0 0 Oa!
40 50 64 66 40 51 63 64 19 3(~ 50 59 0 0 0 0a 0 0 0 0~ 40 50 58 57 13 21 40 37 4 7 17 18
Turbidities were measured on a Klett-Summerson photoelectric colorimeter. Figures are averages of triplicate tubes. b Composition of medium described in text. The iron, copper, and cobalt contents of this medium were 0.21 p.p.m., approx. 0.017 p.p.m., and approx. 0.01 p.p.m., respectively, as determined b y chemical analyses on an ash of the medium constituents. These concentrations were: 3.8, 0.27, and 0.17 ~moles/liter. Inoculum was 0.1 ml. of a 1 : 10,000 dilution of a 24-hr. broth culture. c Oxine and cobalt concentrations were each 1.0 p.p.m, or respectively 6.9 ~M a n d 17 uM. a No g r o w t h occurred in these tubes after 120 hr. of incubation.
CONALBUMIN
AND OXINE
199
at which a decrease in growth rate was observed were 1.7-2.0 ~M. This value is chemically equivalent to an iron concentration of 3.4-4.0 ~M [-1 mole of eonalbumin complexes with 2 moles of iron (6)-]. The value agreed closely with the analytical value for iron in the medium of 3.6 ~M (see Table I). The minimum concentration of oxine which inhibited growth was >2.8 t~M and <4.2 t~M. This concentration was approximately 20 times the theoretical concentration necessary to complex with the cobalt found by chemical analysis and almost twice that required for the total iron, copper, and cobalt (Table I). A combining ratio of 1 mole of oxine to 2 moles of metal ion was employed for these estimatibns (10). A value approximating this ratio was obtained by determining the conCentration of cobalt required to prevent inhibition of growth caused by excess oxine. In such an experiment a cobalt concentration of approximately 6.8 ~M was required for a total oxine concentration of 6.9 ~M or >2.7 and <4.1 ~M of excess oxine. These figures give a calculated combining ratio of > 1.6 and <2.4 moles of cobalt/mole of oxine. None of the following elements prevented inhibition by 6.9 ~M oxine when tested individually at the indicated ~M concentrations: Cu, 16; Mn, 18; Fe, 18; Zn, 15; Mo, 10; and Mg, 41.
Interrelationships of Conalbumin, Oxine, Iron, and Cobalt When conalbumin, oxine, iron, and cobalt were tested as various combinations and at varying concentrations, well-defined and" quantitative relationships were found. In essence, conalbumin and oxine had little or no inhibitory activity when tested together under certain conditions wherein both were inhibitory when tested singly. Thus, this apparent mutual counteraction of inhibitory activities only occurred at concentrations of both conalbumin and oxine which were inhibitory when added alone. The further addition of either or both of the specific metal ions (iron and/or cobalt), in concentrations less than those required to prevent the inhibition normally given by the particular concentration of complexing agent employed, had no effect. However, the addition of either ion in a concentration sufficient to prevent inhibition by its complexing agent also prevented the counteraction of inhibitory activity. Finally, the addition of both metal ions in concentrations sufficient to prevent inhibitions by their complexing agents again allowed for growth.
200
ROBERT
E. FEENEY
The results of one of more than 20 experiments on this subject are presented in Table I. From inspection of the data it can be seen that only slight inhibition (as evidenced only by an increase in the lag period) was obtained with an excess of both conalbumin and oxine (tube sets 3, 4, and 5, Table I). With further addition of either iron or cobalt, inhibition was again obtained; but with further addition of both iron and cobalt, growth occurred (tube sets 4, 5, and 6). These data also show how the counteraction of oxine inhibition was not obtained with an amount of conalbumin which would not at least partially inhibit growth when added alone (tube set 2). Similar quantitative relationships were always found in other experiments. Also, similar quantitative relationships for oxine and cobalt were found. The minimum concentration of oxine necessary to allow for growth when tested with 6.9 tLM conalbumin was the same as that necessary to cause inhibition, >2.8 t~M. The addition of 3.4 pM cobalt or 3.6 ~M iron did not influence the growth given by a mixture of 6.9 t~M conalbumin and 6.9 pM oxine. The above-described, apparently mutually antagonistic activities of conalbumin and oxine did not occur at concentrations 10-20 times greater than the minimum concentrations necessary to give inhibition when tested singly. The concentrations tested were 23 t~M and 46 t~M for conalbumin and 28 ~M for oxine. Prevention of inhibitory activities, however, was given by equivalent concentrations of iron or copper when the complexing agents were tested singly. Studies on the effects of high concentrations of iron and cobalt were attempted but were discontinued because of the formation of visible precipitates. Concentrations of iron up to 72 ~M (approx. 20 times concentration of iron in medium) were tolerated, but a cobalt concentration of 68 ~M (approx. 400 times concentration in medium) was partially inhibitory. A very slight precipitation was caused by this concentration of cobalt. The inhibition was largely prevented by 14 or 28 ~M oxine but not by 72 ~M iron. Lower concentrations of cobalt increased the inhibition given by borderline concentrations of conalbumin (tube set 9, Table I).
Effects of Other Iron-Binding Agents and Proteins Two other substances capable of complexing with iron were found to substitute at least partially for conalbumin in giving the mutual
CONALBUMIN AND OXINE
201
counteraction of inhibitions with oxine. The one, hydroxylamido ovomucoid, an iron-binding derivative of the egg white protein ovomucoid (5), inhibited growth at concentrations > 60 ~g./ml. ; the inhibition was prevented by the addition of iron but not by other metal ions tested. Inhibition was also prevented by the addition of 6.9 ~M oxine; in all other respects studied, the synthetic complexing agent behaved like conalbumin in mixtures with oxine. The other complexing agent, o-phenanthroline, inhibited growth at concentrations > 11 ~M. When it was tested at 22 ~M with 6.9 ~M oxine, a counteraction of inhibitory activities occurred, but only slow and slight growth was obtained. However, this result was considered confirmatory because inhibition by o-phenanthroline was also only poorly prevented by the addition of iron. To test the possibility that a protein per se, or various of its reactive groupings, might nonspecifically have effects similar to those of conalbumin, crystalline ovalbumin was tested. At concentrations of 0.45, 4.5, and 90 t~M, ovalbumin had no effect on the inhibition caused by 6.9 ~M oxine.
Interrelationships When the Conalbumin and Oxine Were Separated by a Dialysis Membrane These experiments were conducted with the dialysis tubes described under the Methods section. In a Preliminary series of eight experiments, these tubes were employed to determine whether or not conalbumin would inhibit growth when the conalbumin was added inside the bag and the medium was inoculated outside the bag. This proved to be the case, ~ and the addition of iron prevented this inhibition. In a further series of four experiments on the interrelationships of oxine and conalbumin, it was found that the counteraction of inhibition in conalbuminoxine mixtures also occurred when the bacteria were separated from the conalbumin (see Table II).
Effects of Other Metal Ions; Media and Cultural Conditions Several factors were found to influence to varying degrees the interrelationships between conalbumin and oxine and the inhibitions given 2 In several earlier experiments, more rapid growth occurred when the organisms were separated from the conalbumin. However, these experiments were discounted
because they were performedwith dialysistubes and bags which had not been soaked in citrate solution.
202
R O B E R T E. F E E N E Y
TABLE II
Expt.
Interrelationships with Organism Separated from Conalbumin by a Dialysis Membrane Relativegrowthr Additions~to medium inside bag Inoculated b after hours indicated 16 24 41 65
A
None Conalbumin Oxine Conalbumin -{- oxine Conalbumin Conalbumin + oxine
Outside bag Do. Do. Do. Inside bag Do.
B
N one Conalbumin Conalbumin -{- oxine
Outside bag Do. Do.
2 0 0 0 0 0
4 0 0 tr 0 1
4 0 0 2 0 2
-0 0 3 tr 3
17
22
41
66
3 0 1
4 0 2
0 4
tr 4
The medium and PreParation of dialysis tubes are described in text. The pH of the medium in these experiments was 8.0. The total volume inside and outside the dialysis bag was 25 ml. The concentrations of conalbumin and oxine added were respectively 4.6 ~M and 6.9 ~M as calculated on total volume of medium. b Outside bag and inside bag refer to site of inoculation. Inocula were 0.1 ml. of a 1 : 10,000 dilution of a 24-hr. broth culture in distilled water. c Growth was estimated visually and reported as tr = trace, and 1 to 4 = gradations from slight to maximum growth. In each case the growth reported refers to media inside or outside of bag corresponding to site of inoculation. The other section always remained sterile. b y t h e s e m a t e r i a l s . E x t e n s i v e effects w e r e o b t a i n e d b y a d d i n g o t h e r m e t a l ions in t h e p r e s e n c e of e i t h e r or b o t h of t h e m e t a l c o m p l e x i n g a g e n t s . C o p p e r i n c r e a s e d i n h i b i t i o n b y c o n a l b u m i n , oxine, or a m i x t u r e of t h e t w o ( t u b e sets 7 a n d 8, T a b l e I ; T a b l e I I I ) . I n o t h e r e x p e r i m e n t s complete inhibition was not obtained with copper, conalbumin, and oxine as in t u b e set 8, T a b l e I . M a n g a n e s e p a r t i a l l y p r e v e n t e d t h e effect of c o p p e r in o x i n e - c o b a l t m i x t u r e s (.Table I I I ) . M o l y b d e n u m a n d m a g n e s i u m h a d l i t t l e effect, while zinc w a s s t r o n g l y i n h i b i t o r y e v e n in t h e a b s e n c e of t h e c o m p l e x i n g a g e n t s . That the conalbumin-oxine system was delicately balanced was s h o w n b y e x p e r i m e n t s on t h e effect of c h a n g i n g t h e size of t h e i n o c u l u m . I n c r e a s i n g t h e size of t h e i n o c u l u m 100-fold i n c r e a s e d t h e c o n c e n t r a t i o n of c o n a l b u m i n r e q u i r e d t o c o m p l e t e l y i n h i b i t g r o w t h f r o m < 4.6 ~ M t o > 6.9 u M b u t , p o s s i b l y a s a d i r e c t c o n s e q u e n c e of t h i s d e c r e a s e in sensit i v i t y of t h e o r g a n i s m t o c o n a l b u m i n , d e c r e a s e d t h e r a t e of g r o w t h in
CONALBUMIN AND OXINE
203
c o n a l b u m i n - o x i n e m i x t u r e s . C h a n g i n g t h e s o u r c e of t h e a n i m a l p r o d u c t c o n s t i t u e n t s of t h e m e d i u m or a d d i n g t h e p h o s p h a t e p r i o r to s t e r i l i z a t i o n also c h a n g e d t h e g r o w t h in c o n a l b u m i n - o x i n e m i x t u r e s .
Comparative Studies with Other Bacteria Several organisms which were inhibited by c o n a l b u m i n by varying d eg r ees w e r e s e l e c t e d for t h e s e studies. All of t h e s e o r g a n i s m s w e r e TABLE III
Effects of Zinc and Manganese on Inhibitions by Conalbumin and Oxine Expt.
A
B
C
Tube set 1 2 3 4 5
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5
Additionsa to medium None Oxine Oxine, Co Oxine, Co, Cu Oxine, Co, Cu, Mn
None Conalbumin Conalbumin, Co Conalbumin, Cu Conalbumin, Co, Conalbumin, Zn, Conalbumin, Co, Conalbumin, Co, Conalbumin, Co, Co, Cu Zn, Mn Co, Cu, Zn, Mn
Cu Mn Cu, Zn, Mn Cu, Zn Cu, Mn
None Conalbumin Conalbumin, Zn Conalbumin, Zn, Co Conalbumin, Zn, Co, Cu, Mn
Turbiditiesbafter hours indicated below 16 24 40 66 35. 0 30 0 19
63 0 47 5 31
76 0 57 22 41
97 0 70 29 50
18
25
42
66
38 16 1 6 0 0 0 0 0 31 2 30
62 36 18 25 0 0 0 0 0 55 31 52
78 54 39 38 3 0 0 0 9 74 68 63
99 79 59 56 47 0 0 0 35 87 78 74
17
25
41
116
40 23 0 0 0
62 45 0 0 0
76 55 0 0 0
--0 0 0
Concentrations of materials added expressed as #moles/liter were: oxine, 6.9; conalbumin (Expt. B), 2.3; conalbumin (Expt. C), 2.0; Co, 17; Cu, 16; Mn, 18; and Zn, 15. b Composition of medium described in text and Table I. Turbidities were measured on a Klett-Summerson photoelectric colorimeter. Figures are averages of triplicate tubes.
204
ROBERT
E.
FEENEY
i n h i b i t e d t o a t l e a s t s o m e e x t e n t on t h i s m e d i u m in t h e p r e s e n c e of a large excess of c o n a l b u m i n ( > 23 ~M). M . lysodeikticus w a s c o m p l e t e l y i n h i b i t e d b y 2.0 ~ M c o n c e n t r a t i o n of c o n a l b u m i n , w h i c h w a s o n l y s l i g h t l y in s t o i c h i o m e t r i c excess of t h e i r o n in t h e m e d i u m a n d signific a n t l y less t h a n t h e c o n c e n t r a t i o n ( a p p r o x . 3.5 ~ M ) r e q u i r e d t o i n h i b i t c o m p l e t e l y M . pyogenes v a r . albus u n d e r t h e s e c o n d i t i o n s . B. subtilis, h o w e v e r , while its g r o w t h r a t e was p r o g r e s s i v e l y r e t a r d e d as t h e c o n c e n t r a t i o n of c o n a l b u m i n w a s i n c r e a s e d a b o v e 2.0 ~ M , r e q u i r e d r e l a t i v e l y h i g h c o n c e n t r a t i o n s ( > 23 ~ M ) for c o m p l e t e i n h i b i t i o n . B o t h of TABLE IV
Interrelationships of Conalbumin, Oxine, Iron, and Cobaltfor Bacillus subtilis and Micrococcus lysodeikticus Tube set
1 2 3 4 5 6 7 8
Additions to medium a
None Conalbumin (1.6) Conalbumin (4.6) Conalbumin (4.6), Fe (21) Oxine (6.9) Oxine (6.9), Co (17) Conalbumin (1.6), oxine (7) Conalbumin (1.6), oxine (6.9), Fe (7)
Growth b of organisms after hours indicated B. subtilis M . lysodeikticus
i6
66
2 2 tr 2 0 2 0 0
4 4 4 4 0 4 0 0
140 4 4 4 4 0 4 1c 0
16 66 1 tr 0 tr 0 tr tr 0
4 3 0 4 0 4 3 0
!40 4 3 0 4 0 4 4 0
Composition of medium described in text and in footnotes of Table I. Figures in parentheses signify concentrations in ~moles/liter of materials added. b Inocula were 0.1 ml. of a saline suspension of a 1 : 10,000 dilution of a 24-hr. broth culture. Growth was estimated visually on duplicate tubes and reported as: tr = trace, and 1 to 4 = gradations from slight growth to maximum. c Good growth in oxine-conalbumin mixtures was obtained in other experiments with B. subtilis where the higher level of conalbumin (4.6 ~M) was employed. t h e s e o r g a n i s m s were i n h i b i t e d c o m p l e t e l y b y 6.9 p M oxine ( T a b l e I V ) a n d s h o w e d t h e m u t u a l c o u n t e r a c t i o n of i n h i b i t o r y a c t i v i t i e s in c o n a l b u m i n - o x i n e m i x t u r e s , b u t in b o t h cases t h e c o n c e n t r a t i o n s of c o n a l b u m i n r e q u i r e d for t h i s c o u n t e r a c t i o n were d i r e c t l y r e l a t e d t o t h e c o n c e n t r a t i o n s r e q u i r e d for i n h i b i t i o n . T h u s , g o o d g r o w t h of M . lysodeikticus o c c u r r e d in c o n a l b u m i n - o x i n e m i x t u r e s a t 1.6 ~ M c o n a l b u m i n ; t h i s w a s l o w e r t h a n t h e c o n c e n t r a t i o n o p t i m u m for M . pyogenes v a r . albus(see T a b l e I). F o u r o t h e r b a c t e r i a s e l e c t e d were g r a m - n e g a t i v e r o d s w h i c h were o n l y s l i g h t l y i n h i b i t e d b y c o n a l b u m i n . A s r e p o r t e d b y R u b b o et al. ( t 0 ) ,
CONALBUMIN AND OXINE
205
a concentration of oxine as high as 27 t~M did not inhibit the gramnegative rods. This concentration of oxine also did not appear to influence any inhibition of these organisms by conalbumin.
Complexing of Copper, Cobalt and Zinc with Conalbumin The formation of complexes of copper, cobalt, and zinc with conalbumin was of obvious interest in these studies. It has been previously reported that copper forms an easily dissociated yellow complex and cobalt does not form a colored complex (6). Results of this same study also indicated that the weak linkage with copper involved the same reactive groups and properties of the protein as the strong linkage with iron involved. In fact, the yellow color of solutions of the copper complex was quickly displaced by the salmon-pink color of the iron complex upon the addition of iron. On the basis of this information, the effects of cobalt and zinc on the development of the copper color were studied in an attempt to determine the combining capacities of conalbumin for cobalt and zinc relative to its weak capacity for copper. Neither cobalt nor zinc gave any color when tested at concentrations of 80-100 ~M with 11.5 t~M conalbumin in phosphate-bicarbonate-citrate buffer (6) at pH 7.6. Similar concentrations in phosphate-bicarbonate buffer at pH 7.6 also did not influence the development of the yellow color upon the addition of 25 ~M copper. The complexing of conalbumin with cobalt or zinc, therefore, appeared to be, at the most, extremely weak. DISCUSSION
The studies of Rubbo et al. (10) on the action of oxine clearly show an important role for iron and copper in the inhibitory action of oxine with gram-positive bacteria. Their results demonstrate the absence of toxicity of oxine on oxine-extracted media, the presence of toxicity of oxine on oxine-extracted media when iron and copper are added, and the absence of oxine toxicity when the oxidation-reduction (redox) potential of the medium is lowered by the addition of ascorbic acid. They support the hypothesis that the toxic effects of oxine are due to the action of lipide-soluble, toxic, oxine complexes of iron or copper per se and not to the free metal ions, The beneficial effects of cobalt on growth under their conditions are attributed to its prevention of the actions of these oxine complexes of iron or copper and not to the prevention of their formation.
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E. F E E N E Y
The results of the present investigation can be interpreted as confirming the dependence of oxine activity on the presence of other trace elements. They can also be interpreted as showing tha t the antibacterial activity of conalbumin is likewise dependent on other trace elements. Such interpretations require the assumption that the absence of inhibitions in mixtures of these materials is caused by their metal-binding properties. The observed quantitative interrelationships support such an assumption rather than some less evident possibility such as chemical interreaction between the conalbumin and oxine. The results are not easily explained, however, on the basis of the formation of toxic complexes as proposed for oxine (10). Conalbumin binds copper so loosely that the complex is dissociated with citrate (6). The toxic complex of oxine with copper advanced by Rubbo et al. should therefore exist in the presence or absence of conalbumin, and conalbumin should not counteract oxine inhibition by binding the copper. Although the addition of copper to conalbumin-oxine mixtures was strongly inhibitory, there should have been ampl e copper in the medium to form a toxic complex with oxine. :While there is no a priori reason why two different metal ion complexing agents should inhibit bacterial growth by similar mechanisms, a comparison of their possible mechanisms of action should aid in understanding these mechanisms. The results with conalbumin were such that its inhibitory activity could not be interpreted on the basis of the formation of a toxic complex. Conalbumin apparently does not form stable complexes with either cobalt or copper and its complexes are not lipide-soluble. Finally, the demonstration that conalbumin inhibits bacterial growth when the protein and bacteria are physically separated by a cellophane membrane eliminates any possibility of a direct action of a complex on the organism. These considerations and results not only necessitate a different hypothesis for conalbumin activity but also strongly suggest that a different hypothesis is necessary for oxine activity. The results of this investigation might be interpreted as further examples of the deleterious effects resulting from imbalances in the concentrations of trace elements. The inhibition of the growth of M. pyogenes var. albus by conalbumin on the medium studied would thus be caused by the binding of iron and the resulting relatively low concentrations of iron and relatively high concentration of cobalt available to the organism. Likewise, the inhibition by oxine would be caused by relatively low concentrations of cobalt and relatively high concentra-
CONALBUMIN
AND OXINE
207
tions of iron. Finally, the absence of inhibitory activities when conalbumin and oxine are added together Would result from the simultaneous lowering of the active concentrations of both iron and cobalt and the consequent absence of an imbalance. The fact that growth does not occur at high levels of conalbumin or oxine in conalbumin-oxine mixtures indicates that a small, but criticai, amount of the active element is necessary and thus favors an imbalance interpretation. Obviously, such a simple interpretation as an imbalance does not explain why the imbalance is toxic. Nevertheless, despite these considerations, the toxic effects of copper and zinc observed in this study are difficult to explain unless one hypothesizes other imbalances involving these ions. The addition of copper or zinc, and possibly other metal ions, would thus cause new imbalances. The studies of Schade (11) on the influence of media constituents on cobalt toxicities further illustrate the manifold factors to be considered in such relationships. In the present study, sulfhydryl groups of media constituents were not considered to play a primary role in the conalbumin-0xine interrelationships. Conalbumin apparently has no sulfhydryl groups, and ovalbumin, at 90 ~M, a concentration sufficient to give approximately 300 ~M sulfhydryl (7), did not affect inhibition by oxine or growth in the absence of oxine. The possibility that chemical interactions involving conalbumin and oxine are responsible for these antagonistic effects has not been eliminated. Unpublished studies of the author in cooperation with S. S. Elberg and W. S. Waring of the University of California indicate some very slow interactions under specific conditions, as previously reported for an excess of o-phenanthroline and the conalbumin-iron complex (6). However, it appears difficult to interpret these effects as mutual inactivation. There are at least two advantages of such a specific metal-binder as conalbumin for studies of this nature. In order to similarly change the concentrations of metal ions by the direct addition of salts to media, it would be necessary to add such high levels that non-specific effects such as precipitations might occur. Another advantage is that the protein or its complexes do not pass through dialyzing membranes; thus intimate contact of complexing agent and organisms is eliminated. The interpretations of these results are important in considering the antibacterial properties of egg white and the role of conalbumin therein
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(12, 13). They indicate that variations in contents of trace metal ions other than iron might strongly influence the antibacterial activity. ACKNOWLEDGMENTS The author gratefully acknowledges the assistance of David A. Nagy throughout this investigation, the performance of the copper and cobalt analyses by E. F. Potter, the preparation of the hydroxylamido ovomucoid by Heinz Fraenkel-Conrat, and the performance of several analyses bY E. D. Ducay. SUMMARY
The growth of three gram-positive organisms was inhibited by addition of either conalbumin or oxine (8-hydroxyquinoline). This inhibition did not occur with further addition of iron in the case of conalbumin, and of cobalt in the case of oxine, or with the addition of both conalbumin and oxine together. When conalbumin and oxine were added together, inhibition was again obtained by adding either cobalt or iron but was not obtained by adding both cobalt and iron. The significance of these findings is discussed. I~EFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. ]0. 11. 12. 13. 14.
ALDERTON, G., AND FEVOLD, H. L., J. Biol. Chem. 164, 1 (1946). ALDERTON,G., WARD, W. H., AND FEVOLD, H. L., Arch. Biochem. 11, 9 (1946). BAIN, 5. A., AND DEUTSCH, H. F., Or. Biol. Chem. 172, 547 (1948). FEENEY, R. E., AND GARIBALDI,J. A., Arch. Biochem. 17, 447 (1948). FRAENKEL-CONRAT,H., Arch. Biochem. 28, 452 (1950). FRAENKEL-CONRAT,H., AND FEENEY, R. E., Arch. Biochem. 29, 101 (1950). MACDONNELL,L. R., SILVA,R. B., AND FEEI~Eu R. E., Arch. Biochem. Biophys. 32, 288 (1951). MCFARLANE,W. D., Biochem. J. 26, 1022 (1932). McNAUGHT, K. J., Analyst 67, 97 (1942). RUBBO, S. D., ALBERT, A., AND GIBSON, M. I., Brit. J. Exptl. Path. 31, 425 (1950). SCHADE,A. L., J. Bact. 58, 811 (1949). SCHADE, A. L., AND CAROLINE, L., Science 100, 12 (1944). SCHADE,A. L., AND CAROLINE, L., Science 104, 340 (1946). WARNER, R. C., AND WEBER, I., Am. Chem. Soe. Abstracts of Papers, p. 24C. l l 9 t h meeting, 1951.