Experimental studies in metal cancerigenesis. V. Interaction of CrIII and CrVI compounds with proteins

Experimental Studies in Metal Cancerigenesis.V. Interaction of CrlI1and Cr” CoxnpoundsWith Proteins Charles H. Grogan and Hans Oppenheimer’ Prom the National Cancer Institute, National Institutes of Health, United States Public Health Service, Department of Health, Education and Welfare, Bethesda, Maryland

Received November 4, 1954 INTRODUCTION

Although chromium appearsto be omnipresent in very minute quant,ities in plants and animals, no demonstrable biological function of the elementhas asyet beenfound. It has beenshownto be widely distributed in the tissuesof man and other mammalsin trace quantities (l), in vegetables and other food products (l-3), in marine products (4), and in river water, subsurface mater (l), and sea water (6). There are also recorded instances of plant diseaseattributed to abnormal chromium concentration in the soil (3,7). As pointed out in a previous paper (8), investigation of certain aspects of the metabolism of chromium was prompted largely becauseof the excessivelyhigh incidence of lung cancer among workers professionally exposedto chromium, its ores, and other chemical compounds; albeit many other and by far more numerous lesions have also been reported from such exposures(9). TOXICOLOGY

After more than a century of investigation of the toxicology of Cr”’ and CrVr compoundsin man (9), animals (N-12), and microorganisms (13), there remain many apparent anomalies to be resolved. The most striking chronic and acute toxic effects have been noted largely with Crvl compounds and have been attributed to the high solubility and diffusibility under physiological conditions, escharotic, and oxidizing 1 Present address: Payne Whitney Clinic, New York Hospital, New York 21, x, Y. 204

METAL CANCERIGENESIS. V

205

properties of CrV’ compounds. CP compounds, with but few exceptions (lo), have been reported largely as having little or no acute toxicity or as being completely innocuous even on longer term administration in massive doses (ll), and have been employed in determining digestibility of diets (14, 15) and as carriers of radioactive phosphorus in viva (16). This apparent innocuousness, at least acutely, of Cr”I compounds has been attributed largely to their insolubility. The arguments regarding a possible chromium etiologic factor in cancer have centered around the water or acid solubility of such an agent (8, 9). CHROMIUM-PROTEIN

INTERACTIONS

Aside from the extensive literature of leather chemistry, studies of the interaction of chromium compounds with proteins have been relatively few. Michael’s (17) studies of the precipitation of proteins with anionic and cationic chromium complexes led him to conclude that the phenomenon was due to salt formation. He found that anionic complexes precipitated proteins on the acid side of the isoelectric point in a manner closely paralleling the acid-combining capacity of the protein in most cases, while cationic complexes precipitated maximally on the alkaline side but utilized only a fraction of the maximal base-combining capacit’y. Quite similar results were obtained by Hara with proteins and cationic, anionic, and neutral complexes of cobalt (l&z) and chromium and other metals (18b, c) at pH’s of 8.8 and 2. He also found that there was no precipitation with neutral or complexes of low charge at either pH. Such stoichiometric relationships have been observed with proteins and acid dyes (19), metaphosphoric acid (20), and chondroitinsulfuric acid (21). Amat (22) reported strong precipitat,ing effect on several proteins, wit,hout giving details of pH or mechanisms, of chromium hydroxide sol at 2 g./l. concentrat,ion. Crystalline derivatives of human serum decanol albumin and other proteins with CrVr have been obtained by Levin (23). Considerable data of qualitative or quasi-quantitative nature of the binding of Cr”’ and CrV1 compounds to proteins have been obtained using CrS1in recent years. Gray and Sterling (24, 25) showed that hexavalent Cr51 is rapidly taken up by erythrocytes in viva and in vitro (Brard (10) earlier pointed out such a selective partition of CrV1 between erythrocytes and plasma proteins in dogs) while CP’ was largely retained and “bound” to plasma proteins. These findings have led to the use of tri- and hexavalent Cr5’ for the determination of circulating red cell volume in man (25, 26), plasma volume in man (27), simultane-

206

C. H. GROGAN AND H. OPPENHEIMER

ous determination of both (28, 29), and determination of erythrocyte survival time (30). Distribution studies of Crs1C18in rats a.nd rabbits led Kraintz and Talmage (31) to conclude that Cr’r’ was “bound” to plasma proteins and proteins at the sites of localization. LEATHER

CHEMISTRY

Chrome tanning in leather production has occasioned the extensive investigation of the interaction of chromium complexes with collagens and gelatins, chiefly at lower pH’s (below 4). Detailed treatment of the theory and technology of chromium complexes and their importance in chrome tanning is presented by Stiasny (32), Herfeld (33), and Grassmann (34). The possible mechanisms involved in the bonding of chromium to proteins are reviewed by Gustavson (35). Earlier theories are discussed by Gustavson (36) and Shuttleworth (37, 38). Gustavson demonstrated that anionic and neutral as well as cationic chromium complexes were bound by collagen (3940). The importance of carboxyl groups in the fixation of chromium by proteins was shown by Bowes and Kenten (41). When these groups were esterified, fixation decreased. Green (42) showed that amino and hydroxyl groups, as well as carboxyl groups, along the peptide chain enter into the fixation of chromium. Varying degrees of acetylation of free amino or hydroxyl groups of collagen decreasedfixation of either cationic or anionic chromium complexes. Strakhov (43) showed that collagen fixed more chromium than silk fibroin (less free amino and carboxyl groups) and that an e-caprolactam polymer, devoid of carboxyl and amino groups, fixed none. Present evidence indicates the initial formation of a salt link with carboxyl groups which is further stabilized by penetration of the protein carboxyl group into the coordination sphere of chromium displacing another ligand. It is probable that the same type of change takes place once an anionic complex becomes initially attached to a basic protein group. Additional stability of the chromium-protein complex is then provided by further coordination of sterically available hydroxyl or amino groups on the same or neighboring peptide chains. While theories developed in the field of leather chemistry furnish valuable information relative to the nature of the chromium-protein bonding, the unique structural nature of the solid substrate lends itself to a greater degree of fixation than would be expected for the same type of chemical structure in solution. Such geometric factors may, on the

METAL

CANCERIGENESIS.

other hand, again become important structural protein in z&o.

207

V

in depot fixation

of chromium to

EXPERIMENTAL

Chromium Solutions. Stock solutions of potassium dichromate, potassium chromate, chromic acetate, and chromic chloride were either the same as previously employed (8) or were freshly prepared from the same reagent samples. Bu$ers. Sodium acetate-acetic acid and sodium and potassium phosphate buffers (8) were used to cover the pH range 4-8. The ionic strength was usually 0.10; but some zone electrophoresis experiments were carried out in the range p = 0.050.17 to study the effects of ionic strength on the separation of components. Dialysis Membranes. Visking seamless clear cellophane tubing of various diameters was used. Protein Solutions. It was desired to first establish the degree of over-all binding of Cr”’ and CrvI compounds to human plasma proteins as a whole and ultimately to differentiate as to the degree and specificity of binding, if any, between individual plasma proteins. Two approaches were apparent: the one to study each human plasma protein individually without quantitative knowledge of over-all binding and the other to ascertain the extent of binding by all plasma proteins and attempt to separate the individual components and bound chromium by electrophoretic or other techniques. The latter approach was adopted for the initial experiments. Because of the complex behavior of CF compounds in aqueous media (8), pilot experiments were carried out with soluble egg-white proteins which served as a suitable protein mixture to test the various techniques employed. Solutions of nominal 10% protein concentration were prepared from the solid protein mixture in the desired buffer, and the pH was adjusted with dilute sodium hydroxide or hydrochloric acid and filtered through a Seitz clarifying filter and a sterilizing filter. Solution not used immediately was stored in sealed ampoules at 5°C. Pooled “normal” human plasma was obtained from the Public Health Service Hospital, Baltimore, Maryland, courtesy Dr. Thomas Crahan, and the Georgetown University Hospit)al, Washington, D. C., courtesy Dr. B. H. Armbrecht. Human plasma samples were also obtained from individual donors. The only additive used in collected the human plasma samples was 4-5 mg. heparin sodium/50 ml. blood.

Methods Analytical. Microdeterminations of chromium in the various media employed were performed by procedures to be reported separately.* Nitrogen determinations on the protein solutions were performed in the Institutes’ microanalytical laboratory under the direction of Dr. W. C. Alford. Test Tube Interaction Experiments. Preliminary data on the initial and long 2 C. H. Grogan,

H. J. Cahnmann,

and E. Lethco,

unpublished

results.

206

C. H.

GROGAN

AND

H.

OPPENHEIMER

contact interaction of Crlrl and Cry* with egg albumin were obtained by adding chromic acetate or potassium chromate (several increments within the range MO1000 pg. Cr/ml.) to 5-ml. portions of 10% (exact nitrogen content determined by micro-Kjeldahl) sterile protein solutions at 5°C. and pH’s of 4.14, 5.30, 6.40, and 7.35. The samples were stored at 5°C. and observed periodically for 30 days, opened, purposely contaminated by exposure to the atmosphere, resealed, and observed for 21 months. Information on the precipitation by Crrrr and Crvr, reduction of Crvl on long contact with protein, and self-sterilization of the chromium-protein solutions at various pH’s was obtained. Dialysis Experiments. Studies of the binding of Crrrr and Crvr to egg albumin and human plasma by equilibrium dialysis and modified equilibrium dialysis techniques were carried out employing the two dialysis arrangements previously described (8). Egg albumin solutions at each pH (5-10 ml.) were equilibrated against 100-125 ml. of buffer at 5°C. for 24 hr. and then inserted in fresh buffer. The chromium solution was then added either to the protein solution or to the dialyzate buffer. Since the results are expressed in terms of micrograms Cr bound/mg. protein nitrogen, preliminary dialysis of the plasma against one to three changes of 125 ml. of buffer eras necessary to remove diffusible components. The number of buffer changes required at each plasma dilution was determined by nitrogen determination in successive dialyzates by Kjeldahl digestion and nesslerization. Hexavalent Chromium. Since chromate was found to be completely diffusible throughout the pH and concentration range investigated (8)) equilibrium dialysis binding studies were uncomplicated. In these experiments the Crvr concentrations at each pH were kept below the level at which precipitation occurred as determined in the test-tube experiments that had indicated greatest interaction at low pH’s as the protein became more positive in charge. The rate of apperance of Crvl in the dialyzate was studied as a function of pH; 400-460 fig. Crvl/ml. was added to 5-6 ml. of egg albumin (9-10 mg. N/ml.) in the 125-ml. dialyzer, and 109pl. dialyzate aliquots were analyzed periodically until a steady Crvr concentration was attained. CrVr content inside and outside the membrane, and nitrogen concentration were then determined. Identical experiments were performed in which the first dialyzate was discarded after a maximum Crvr concentration was attained, and the rate of CrYr appearance in fresh buffer was studied to ascertain the relative strength of binding at each pH. Equilibrium dialysis experiments were run with egg albumin in the 30-ml. dialyzer. Ten milliliters of a nominal 10% egg albumin solution, previously equilibrated against buffer, was placed into the dialysis tube containing lo-12 ml. of buffer, chromate solution added, the whole sealed, dialysis continued for 48 hr., and the concentration of Crvr inside and outside and nitrogen inside the membrane determined. Separate pairs of experiments were run in which Crvr was added inside and outside the membrane and the protein solution diluted to 35, 34, Ji, or $g of its initial concentration until a maximal ratio of bound Crvr was obtained. Dilution of the protein solution in the presence of a constant amount of Crvr increased the molar ratio of CPr to protein and decreased the Donnan effect. Dialysis experiments with Crvr and pooled “normal” human plasma and several plasma samples from individual donors were carried out in this manner at pH 7.35.

METAL

CANCERIGENESIS.

V

209

l’riualent Chromium. The use of dialysis techniques in the study of the binding of Cr*I* to proteins is complicated by the dependence of the diffusibility of CG complexes formed from chromic acetate in p = 0.1 acetate and phosphate buffers on Cr”I concentration, pH, and age of the solution. Initial studies of the rate of appearance of CrlIr in the dialyeate, analogous to those performed with Crvl and egg albumin, indicated increased binding of Cr”* with increasing pH (4-7.35). These observations were largely fortuitous, however, as dialysis of chromic acetate from buffer solutions under identical conditions showed increased retention of CG1 within the membrane with increasing pH. Since we were primarily interested in pH conditions near those of blood, efforts were centered on pH 7.35. Dialysis of chromic acetate aged various times in contact with egg albumin showed that more CrI*I was retained at all times of aging in the protein solution than in pH 7.35 phosphate buffer under identical conditions (cf. Ref. (8), Fig. 3, and Discussion). This difference could not be unequivocally attributed to bound Cr”’ because of the unknown effects of the protein solutions on the formation, stabilization, and diffusion of elate polynuclear Cr”I complexes. Previous studies (8) had shown that Js or more of the Cr”I (1O-4-1O-8 M) complexes formed in phosphate buffer, pH 7.35, were diffusible after 60 days aging at 5°C. and that certain concentrations of chromic acetate (below 1O-4 M) reached equilibrium in the system when stirred on both sides of the membrane. Based on these observations, experiments were carried out in the 125-ml. dialyzer comparing the concentration of Cr”I attained in protein solutions and buffer “blanks” when chromic acetate was allowed to age various lengths of time in the buffer prior to a 24-11%hr. dialysis into the solution within the membrane.

Electrophoresa’s Experiments Solutions of egg albumin and human plasma in ,U = 0.1 phosphate buffer, pH 7.35, to which varying amounts of chromic acetate or potassium chromate in the range IO-LlO-* M had been added, were subjected to electrophoretic separation by moving-boundary and zone-electrophoresis techniques using essentially the same apparatus and experimental conditions previously outlined (8). Some zone electrophoretic separations were made at p = 0.05 to obtain better resolution. The effects on the distribution, mobility, and chromium content of the components were investigated for protein and chromium solutions (a) aged together and (6) aged separately before electrophoresis. Such mixtures were subjected to zone electrophoresis directly. In the moving-boundary experiments, preparatory dialysis is employed for equilibration with respect to T, pH, and F. The patterns obtained by aging the chromium and protein together prior to dialysis were compared with those obtained on mixing the separately aged chromium solution with the separately dialyzed protein. The protein and lipide components were located on the strips by the’ dyeing techniques described by Durrum et al. (44) and their relative concentrations determined by means of a photoelectric densitometer. Chromium content was determined by cutting up the strips and analyzing for chromium. The moving-boundary patterns were photographed, enlarged, and traced on white paper, and the mobilities and composition were calculated from the areas determined with a compensating polar planimeter.

-

-

P D P D P D P D P’ P

P D

~-

1

-

- -

R P’l.6

R ‘IR P’O. 4

R

R

R

2

CrW

-

R P’1.7

R T R T R T R P’O. 4

R T

3

no precipitation on addition precipitation on addition precipitate rediesolves on mixing precipitation occur8 or recurs on storage overnight or longer/volume in ml.

-

N

956

-

N

523

N, P, D, P’,

N

421

a Symbols:

N

326

T V

T V T V T V T V

T V T V T V T V

N

221

T V -

T V

-

3

T V

-

2

-

N

1

-

CP’

113

+ 0.07670 ma%.

pg.lml.

or

Cr”‘ CP

Added

pH 4.14, 9.88 mg. N/ml.

Protein

at 5”

R, M, T, V,

-

N N

R R P “0.2

-

N

N

R

R P’O. 7

N

R

1

N

-

I-

R

3

-I1

-

/

=

Studies’

T v HO. 1 T V T V T V T V SfO.l T V

-

2

CT ___-

B Ml.0

T v MO.2 T B B MO.2 T B B M4.0

_-

3

N

N

N

N

N

N

1

-

-

I “0.1

T

T

T

T

L

-

B, 1, 2, 3.

-

-I--

3

/ 1

T B IN v M r2.01 co.1 I T ; iN V B (N T V T B 1-J V I T B/N V ! T B ,N I V

2

pH 7.35, 10.04 mg.

T

-

T

-

T

3

-

-

T

T

-

-

- -

T

2

N/ml.

solution blue or blue-green initial observations after 3 months at 5” after 21 months at 5”

R YO.2

T R T R T R R “0.2

T R

T

3

I-

-

2

CIV’

pH 6.40, 10.04 mg. N/ml.

Reduction of Cr”’ to Cr”’ visible mioroorgmiam growth/volume in ml. trace of precipitate present. less than 0.02 ml. solution still violet or gray-violet

2

CrV’

pH 5.30, 9.32 mg. N/ml.

1

I Interaction

egg albumin, 5’3.

Aging

TABLE Five milliliters

Chromium

METAL RESULTS

CANCERIGENESIS.

211

V

AND DISCUSSION

The results of the test-tube experiments are shown in Table I. No visible reaction occurred with CrIII, and the solutions were still violet (below pH 6.4) without precipitation at the maximum time. The amount of precipitate with CrVT was greatest at, the lowest pH and paralleled the degree of binding found in dialysis experiments. Visible reduction of CrV1 in long contact with protein solutions took place up to pH 6.4. CrV’ solutions were self-sterilizing at all pH’s, while molds grew readily in Cr’I’ solutions above pH 6. Table II summarizes the dialysis experiments wit,h egg albumin and CrV1. ‘The amount of CrV1 bound decreased with increasing pH until at pH 8.10 it was negligible (within experimental error). All CrV’ could be dialyzed away from the protein at pH 7 and above by changing the dialyzate once. The rate curves, Fig. 1, show the increasing rate of appearance of CrV’ in the dialyzate and decreasing magnitude of binding with increasing pH. Table III summarizes the results of dialysis experiments with egg albumin and chromic acetate. Part A shows typical results obtained by aging 8.6 X 1O-3 2cI Cr”’ with and dialysis away from egg albumin at TABLE

II

Dialysis Experiments, Egg AlbuminCr” PH

4.14 5.30 6.40 7.35 8.10

24-28 hr.

Preliminary dialyses 1st buffer 24 hr. 2nd buffer pg. Cr/mg. N

12.5 8.0 7.7 4.2 3.4 0.55 -

8.8 9.3 1.48 1.36 0.72 ca. 0 -

Equilibrium dialyses pg. Cr/mg. N”

6.9

f

.3*

1.80 f

.lO=

0.63 f

.I3

0.44 f 0.08 f

.ll O.Ofid

@These values represent the highest ratios of Crvl to protein obtained in the series of equilibrium dialyses in which the protein solutions were successively diluted. ) Obtained at 2% protein level. If a plot of pg. Cr/mg. N vs mg. N is extrapolated to zero (ratio of Cr to protein approaches infinity) this value approaches 8.4 as a limit. c At pH 5.20. d This value is probably within experimental error. The several series of experiments at this pH gave small positive values in the range 0.03-0.13.

212

C. H.

GROGAN

AND

H.

OPPENHEIMER

-E

I

I

20

IO TIME

30 OF

DIALYSIS

40

50

t

HOURS

FIG. 1. Rate of exit of Cry’ from egg albumin solutions. p = 0.1; temp. = 5O; E = equilibrium concentration.

pH’s. The apparent binding increases with pH. However, an analysis of the chromium content of the protein solution and dialyzate does not give a true picture of bound CP under these conditions due to the presence of nondiffusible Cr”’ complexes (8) and the unknown effect of the protein on the formation and diffusion of such complexes. It is probable that a large percentage of the “bound” WI1 reported by Gray and Sterling (24), on the basis of dialyzing CrS1C13away from bovine serum albumin just below the precipitat,ion point, of chromic hydroxide, is likewise due t*o nondiffusible Cr II* hydrolysis and olation complexes formed under these conditions. Figure 2 shows the results of dialysis of 4.5 X 10e3 M CP from protein and buffer solut,ions. More CP was various

METAL

CANCERIGENESIS.

TABLE

III

Dialysis Experiments, Egg Albumin-0”’ A: Aged 30 days pg. Crjmg. N PH

It4

4.14 5.30 6.40 7.35

6.1 9.3 15.5 19.4

213

V

248

372

4.8 9.1 14.1 16.3

3.9 -

-

-

4d

Fresh&*

2.3 -

3.2 3.6 5.6 6.4

B: pH 7.35 Age hr.

1 24 72 170 720 1’540

rrg.Crjmg. 1. Dialysis

A24

3.6 2.0 3.8 4.4 2.3 1.6

N

outward

2. Dialysis

inward

B48

A24

B48

C7ZC

3.5 2.4 3.9 5.1 2.7 2.1

0.22 0.30 0.34 0.74 -

0.77 1.24 1.06 1.18 -

1.08 0.82? 1.10 1.29 -

a Arabic numbers indicate successive fresh loo-ml. portions of buffer; subscript total dialysis time in hours. b Aged 1 hr. or less, dialysis started immediately. c Letters refer to separate experiments each dialyzed for the times indicated.

retained in the protein solutions under all conditions studied, and amounts of “bound” Cr”’ calculated from these curves agreed with the values found in preliminary dialysis experiments, Table III, part A. Table III, part Bl, shows the effect on apparent ‘
214

C. H. GROGAN

AND H. OPPENHEIMER

su8scRIFT

I

I IO

, 20 TIME

FIQ. 2.

I 30 OF AGING

I 40

PI PROTEIN a= BUFFER - DIACYSIS TIME, HOURS

I 50

I 60

DAYS

Dialysis of Crrrr from egg albumin and buffer solution. fi = 0.1; temp. = 5“; pH = 7.35.

noticeably lipemic plasma bound less chromium than did clear nonlipemic samples. Representative data obtained by moving-boundary electrophoresis are shown in Table V. None of the conditions employed with either CI” or CrV’ significantly altered the electrophoresis pattern of eggwhite proteins such as noted by Lessiau et al. (45) with copper and equine serum proteins or Foster and Yang (46) with cationic detergents ovalbumin, and bovine serum albumin. CrV1 had no effect on the electrophoresis patterns of human plasma. Cr”’ did not significantly alter the patterns or mobilities of human plasma protein components except at the higher concentrations when a small, new fast peak, migrating ahead of the albumin, appeared. Investigation of this new component showed that it was not a polynuclear chromium aggregate or heparin, which was present to less than 0.01%. It has not been identified. Similar peaks of unknown nature, migrating

METAL

CANCERIGENESIS.

215

V

0 =BUFFER P = PROTEIN SUBSCRIPT-DIALYSIS

20

I 50

TlME FIG.

3. Dialysis of WI*

1 100 AGING, HOURS

OF

Plasma

IV

Plus CT”’

and CT”, W.S.Cr/w.

Pooled Pooled Donor Donor Donor Donor

1 2 1 2 3 4

pH Y.&i .v CrvIa

CW

Plasma

I 150

into egg albumin. h = 0.1; temp. = 5”; pH = 7.35.

TABLE Human

TIME, nOUR3

1.23 i

0.05*

1.37 f

0.03

1.12 f

0.05

1.54 f 1.36 f 1.71 f

0.08 0.03

1.10 i 0.05 1.16 f 0.06 0.57 f 0.10 1.71 i

0.06

1.31 f -

0.10 0.10

a CrIlI dialyzed into protein 72-100 hr.; CryI dialyzed into and away from protein 24-48 hr. 6 Values represent maximal value of chromium bound at the various plasma dilutions.

216

C.

H.

GROGAN

AND

H.

OPPENHEIMER

ahead of albumin have been reported in cerebrospinal fluid (47) and plasma containing various anticoagulants (48). It was likewise not possible to demonstrate binding of CrV1 to egg albumin or human plasma proteins at pH 7.35 by paper electrophoresis. Experiments at various chromium levels showed no amount of CrV’ significantly greater than that retained by the paper “blank” (8). The protein components were located on one strip and compared with t’he CrV1 content of a replicate strip. At pH 4, where strong binding to egg albumin was indicated by dialysis experiments, and Crvl and protein TABLE Moving-Boundary

V

of Egg-White Proteins and Cr” and CT” buffer, nH _ 7.3-7.4, 3°C.. M = 0.1. =

Electrophoresis Phosphate

zzvz=z=

Absolute

mobilities cm./v./sec.

X 106 sq.

I; lelative

albumin

mobilitie! i, R elative concen= 1.00 tration

-_

Conditions Ascending

Descendin

g

p

wending

1Descending

1. Fresh, dilution from 1070, dialysis2.5days, no Cr 2. Fresh, 10e3 M CrrI1 added to 1070, diluted, dialysis 2.5 days 3. 1O-3 M Cr”I aged in for 3 days, 10% stored 2 days, dilution, dialysis overnight 4. lop3 M Crlll aged 7 days in 1070, dilution, dialysis 2.5 days 5. Same as 4, aged 28 days, dialysis 3 days

6. 1O-3 M Cr”’ aged 5 months, dilution, dialysis 2 days

A scending

Desending

--

--

-7.69 -6.91 -4.37 -2.59 -7.46 -6.71 -4.27 -2.58 -7.59 -6.87 -4.58 -2.76

-6.88 -5.92 -3.54 -2.03 -6.36 -5.35 -3.12 -1.81 -7.02 -6.23 -4.26 -2.50

1.00 0.90 0.57 0.34 1.00 0.90 0.57 0.35 1.00 0.91 0.60 0.36

-7.51 -6.80 -4.30 -2.59 -7.40 -6.64 -4.43 -2.62 -7.51 -6.61 -4.38 -2.39

-7.10 -6.10 -3.55 -2.05 -6.89 -6.01 -3.68 -2.04 -7.16 -5.91 -3.76 -2.22

1.00 0.90 0.57 0.34 1.00 0.90 0.60 0.35 1.00 0.88 0.58 0.32

%

%

0.86 0.51 0.30 1.00 0.84 0.49 0.29 1.00 0.89 0.61 0.36

61 17.5 15 6.5 59 15 15 11 ,60.5 14.5 12.5 12.5

52.5 13 24.5 10 50.5 14 23.5 12 66.5

1.00 0.86 0.50 0.29 1.00 0.87 0.53 0.30 1.00 0.83 0.52 0.31

57.5 15 16 11.5 55 19 17.5 8.5 64.5 10 15 10.5

48 19 20 13 50.5 17.5 23 9 57 10.5 20 12.5

1.00

23.5 10

TABLE Moving

V-Continued

Boundary Electrophoresis of Human Plasma and CrJ1’a Phosphate buffer, pH 7.3-7.4, 3”C., p = 0.1.

-

CrI I I molarity

I

Absolute mobilities X 105 sq. cm./v./sec. Ascending

~-

klative mobilities ,R elative concenalbumin = 1.00 tration

-

-

A,scend-

Descending

ing

kscending

scending %

0

1.83 x 10-d

5.64 X 1O-4

2.26 X 10-a

3.53 x 10-a

5.64 X lo-”

-

-6.09 -5.00 -4.03 -3.49 -2.15 -0.94 -5.99 -4.89 -4.03 -3.53 -2.02 -0.70 -6.09 -4.97 -3.94 -3.52 -2.17 -0.84 -5.99 -4.84 -4.02 -3.42 -2.04 -0.75 -7.07 -6.08 -4.97 -4.01 -3.50 -2.05 -0.82 -6.59 -6.07 -5.00 -4.16 -3.46 -2.06 -0.80

Alb. 01 (12 p Fibr. y

-5.29 -4.45 -3.70 -2.81 -1.87 -0.55 -5.38 -4.63 -3.89 -2.90 -2.01 -0.47 -5.43 -4.64 -3.98 -3.00 -2.06 -0.55 Boundar! disturbed

-

r

(-8.501) -5.41 -4.58 -3.91 -3.04 -1.98 -0.58 -5.25 -4.45 -3.80 -2.86 -2.01 -0.52 -

1.00 0.82 0.66 0.57 0.35 0.15 1.00 0.82 0.67 0.59 0.34 0.12 1.00 0.82 0.65 0.58 0.36 0.14 1.00 0.81 0.67 0.57 0.34 0.13 1.16 1.00 0.82 0.66 0.57 0.34 0.14 1.09 1.00 0.82 0.69 0.57 0.34 0.13

-

1.00 64.5 0.84 0.70 0.53 0.35 0.10 1.00 67 0.86 0.72 0.54 0.37 0.09 1.00 69 0.85 0.73 0.55 0.38 0.10 62 (1.572 ) 3.5 58 1.00 0.85 0.72 0.56 0.37 0.11 6 58.5 1.00 0.85 0.72 0.54 0.38 0.10

Des-

:nding

% 66 5.5 6 8.5 4 10 65 4.5 6 8 5.5 11 63 5.5 6 10 3.5 12 3.5 59.5 6 7 8 5.5 10.5 6 52.5 5 7.5 10 4.5 14.5

0 The human plasma samples were dialyzed against buffer, diluted with an equal volume of buffer, the requisite amount of Cr”’ added, and electrophoresis tarted on an aliquot of this solution 1-l .5 hr. thereafter. 217

218

C. H.

I

6

GROGAN

I

AND

I

I

4 2 DISTANCE

FIG. 4. Paper electrophoresis

H.

0 OF

OPPENHEIMER

I

I

2 4 MIGRATION,

I

6 CM.

of egg albumin plus K$304

I

6

3 t'

. pH = 4.0.

components migrate in opposite directions, a large excess of CrV1 remained with the proteins. A typical diagram is shown in Fig. 4. The lesser migration at this pH, due to the proximity to the isoelectric points of the major components could be partially overcome by increasing the time of electrophoresis. CrV1 was associated with all components and did not appear to be preferentially bound by any individual component. Freshly prepared Cr”’ solutions on aging with egg albumin l-2 days at 5”C., pH 7.35, gave electrophoresis patterns essentially the same as that of the protein alone and Cr II1 distributions similar to Cr”’ solution blanks. Aging the Cr”I- protein solutions for periods up to 41 days resulted in a progressive shift of CrlI1 into the protein bands of the pattern. This suggested an increased binding on long contact. Such a tentative conclusion was further indicated by series of experiments in which Cr”’ and protein solutions were aged separately and together before electrophoresis. A typical pattern obtained in this type of experiment is shown in Fig. 5. When aged separately the Cr”’ distribution closely approximated that from Crxl’ solutions alone. The aged mixtures all showed large shifts of Cr”’ into the regions containing the protein bands. No significant alterations were found in either the electrophoretic protein or Cr II1 distribution patterns of human plasma aged for short times with Crlll or aged separately for longer times. Thus human plasma behaved similarly to egg albumin in this respect. Again, increased aging

219

METAL CANCERIGENESIS. V

-8

6

4

i!

DISTANCE

0 OF

2 4 MIGRATION,

6 CM.

8

IO t

FIQ. 5. Paper electrophoresis of chromic acetate plus egg albumin. pH = 7.35; p = 0.05.

of the components in mixture, but not separately, caused an increasing concentration of Cr’I’ in the protein regions of the patterns. SUMMARY

The binding of Cr”’ and CrV’, as represented by chromic acetate and potassium chromate, to egg proteins and human plasma proteins has been investigated by dialysis and electrophoretic techniques. CrVx was shown by dialysis and by electrophoretic and precipitation experiments to be bound by egg albumin in decreasing amounts as the pH changed from 4 to 7.35. Pooled and individual human plasma samples bound two to three times as much CrV1 at pH 7.35 as did egg albumin. The binding of CrV1 to egg albumin at pH 4 could be demonstrated by paper electrophoresis but could not be demonstrated by this technique at pH 7.35 with either egg albumin or human plasma. The results suggest an essentially electrostatic type of binding [cf. (Refs. 17, 18)j increasing as the positive charge on the proteins increases. Solutions of protein containing from 100 to 1000 pg. Crvl/ml. were self-sterilizing from pH 4 to 8. Reduction of CrV’ occurred on long contact with sterile protein solutions up to at least pH 6.4.

220

C. H. GROGAN

AND

H. OPPENHEIMER

CF, which in the form of chromic acetate at pH 7.35 exists in positive, negative, and neutral complexes of low charge (8), was bound by both human plasma and egg albumin. The complexes formed from chromic acetate at all pH’s studied produced no visible reaction with any of the protein components [cf. (B)] an d were not self-sterilizing at the higher pH’s. Difficulties due to hydrolysis, olation and aggregat’ion to form polynuclear CrI” complexes in aqueous solutions of Cr”’ salts are discussed and data presented showing the effects of these changes on apparent binding as evaluated by dialysis techniques. Conditions permitting differentiation between bound Crnl and nondiffusible Crl” are presented. Dialysis data indicated a decreased binding of chromium in lipemic plasmas in the limited number of cases studied. At pH 7.35, neither CrI’I nor CrV1 in the concentration range lW410e2 M significantly altered the electrophoretic patterns of egg elbumin components. CrV’ under similar conditions produced no significant effect on human plasma proteins. Cr II1 likewise had no effect at the lower concentrations; but at the higher concentration levels produced a small amount of a new component of unknown nature migrating ahead of human serum albumin. Paper electrophoresis at pH 7.35 and Jo = 0.05 indicated binding of Cr”r to human plasma proteins and egg albumin. The binding appeared to increase with time of contact. REFERENCES 1. GRUSHKO, YA, M., Biokhimiya 13, 124 (1948). 2. ST. RAT, L., AND BERTRAND, G., Compt. rend. 337,150 (1948). 3. MONIER-WILLIAMS, G. W., “Trace Elements in Food,” Chap. 20. John Wiley, New York, 1949. 4. NEWELL, J. M., AND MACCOLLUM, E. V., U. S. Bur. Fisheries, Invest. Rept. 6, 1 (1931). 5. DAVIDSON, A. M. M., AND MITCHELL, R. L., J. Sot. Chem. Ind. (London) 69, 213, 232 (1940). 6. ISHIBASWI, M., AND SHIGEMATSU, T., Bull. Inst. Chem. Research, Kyoto Univ. 33, 59 (1950). 7. VAN DER MERWE, A. J., AND ANDERSSEN, F. G., Farming in S. Africa 12,439 (1937). 8. GROGAN, C. H., AND OPPENHEIMER, H., J. Am. Chem. Sot. 77,152 (1955). 9. U. S. Public Health Service Publ. No. 192 (1953). du Chrome,” 10. BRARD, D., “Toxicologic Actualit& Scientifiques et Industrielles No. 228. Hermann et Cie, Paris, 1935. 11. AKATSUKA, K., AND FAIRHALL, L. T., J. Znd. Hyg. 16, 1 (1934). 12. GRUSHKO, YA. M., Farmakol. i Toksikol. 16, (I$, 54 (1953).

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13. INGOLS, R. S., AND KIRKPATRICK, F. S., Anal. Chem. 24, 1881 (1952). 14. SCH~~RCH, A. F., LLOYD, L. E., AND CRAMPTON, E. W., J. Nutrition 41, 629 (1950). 15. KANE, E. A., JACOBSON, W. C., AND MOORE, L. A., J. Nutrition 4’7,263 (1952). 16. GABRIELI, E. R., Acta Physiol. Scand. 33, 283 (1951); Acta Pathol. Microbial. Stand. 31, 195 (1952). 17. MICHAEL, S. E., Biochem. J. 33, 924 (1939). 18. H.IIRA, R., Bull. Chem. Sot. Japan 22, 109 (1949). HARA, R., Bull. Chem. Sot. *Japan 23, 7 (1950); J. Pharm. Sot. Japan 72, 748 (1952). 19. CIIAPMAN, L. M., GREENBERG, D. M., AND SCHMIDT, C. L. A., J. Biol. Chem. 72, 707 (1927). 20. HERMANN, H., AND PERLMANN, G., Nature 140, 807 (1937); Biochem. J. 32, 926 (1938). 21. MEYER, K., PALMER, J. W., AND SMYTH, E. M., J. Biol. Chem. 119,501 (1937). 22. AMAT, M., Rev. real acad. cienc. exact., fti y nut. Madrid 36, 62 (1942). 23. LE:VIN, J., J. Am. Chem. Sot. 73, 3906 (1951). 24. GRAY, S. J., AND STERLING, K., J. Clin. Invest. 29, 1604 (1950). 25. GRAY, S. J., AND STERLING, K., Science 112,179 (1950). 26. STERLING, K., AND GRAY, S. J., J. C&n. Invest. 29, 1614 (1950). 27. FRANK, H., AND GRAY, S. J., J. C&n. Invest. 32, 991 (1953). 28. GRAY, S. J., AND FRANK, H., J. Clin. Invest. 32, 1000 (1953). 29. GRAY, S. J., AND FRANK, H., J. Clin. Invest. 32,571 (1953). 30. EBAUGH, F. G., JR., EMERSON, C. P., AND Ross, J. F., J. Clin. Invest. 32,1269 (1953). 31. KBAINTZ, L., AND TALMAGE, R. V., Proc. Sot. Exptl. Biol. Med. 81,490 (1952). Steinkopff, Dresden and Leipzig, 1931. 32. STYASNY, E., “Gerbereichemie.” der Lederherstellung.” Steinkopff, Dresden and 33. HERFELD, H., “Grundlagen Leipzig, 1950. der Gerbereichemie.” Springer, Vienna, 1939. 34. GRASSMANN, W., “Handbuch 35. GUSTAVSON, K. H., Advances in Protein Chem. 6,353 (1949). 36. GUSTAVSON, K. H., J. Phys. & CoZZoid Chem. 61, 1181 (1947). 37. SHUTTLEWORTH, S. G., J. Sot. Leather Trades’ Chemists 34,410 (1950). 38. SHUTTLEWORTH, S. G., J. Am. Leather Chemists’ Assoc. 47, 387 (1952). 39. GUSTAVSON, K. H., J. Sot. Leather Trades’ Chemists 36, 182 (1952); 34, 259 (1950). 40. GUSTAVSON, K. H., J. Am. Chem. Sot. 48, 2963 (1926). 41. BOWES, H., AND KENTEN, R. H., Biochem. J. 43, 358 (1948); 44, 142 (1949). 42. GREEN, R. W. Biochem. J. 54, 187 (1953). 43. STBAKHOV, I. P., Zhur. Priklad. Khim. 24,142 (1951); English translation, ibid. 24, 159 (1951). 44. DURRUM, E. L., PAUL, M. H., AND SMITH, E. R. B., Science 116, 428 (1952). 45. LESSIAU, J., VIOLLIER, G., AND MACHEBOEUF, M., Helv. Physiol. et Pharmacol. A eta 0, C25 (1948). 46. FOSTER, J. F., AND YANG, J. T., J. Am. Chem. Sot. 76, 1015 (1954). 47. Ho~H, H., CHANUTIN, A., AND PANKEY, T., JR., Proc. Sot. Exptl. Biol. Med. 81, 628 (1952). 38. HOCH, H., AND CHANUTIN, A., .I. Biol. Chem. 197, 503 (1952); 290, 241 (1953).