Hemolysis by colloidal electrolytes

HEMOLYSIS BY COLLOIDAL ELECTROLYTES 1' 2

Sydney Ross and Arthur M. Silverstein 3

Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York

Received February 10, 1954

ABSTRACT

The hemolytic action was measured at 20°C. and at 37°C., of each of a homologous series of sodium alkyl sulfates (C8 to C,8) and of each of a homologous series of benzyi- dimethyla]kylammonium chlorides (C8 to C18) dissolved in water, and in n-propanol- water mixtures, containing 0.05 M phosphate buffer (pH = 7.2), made isotonic with sodium chloride. The results are examined for their bearing on a recent theory of the physical chemistry of hemolysis by surface-active agents, advanced by Pethica and Schulman. The effect of colloidal association on both hemolytic and bactericidal action of surface-active agents is discussed.

INTRODUCTIO ..N-

A series of valuable investigations of the mechanism of hemolysis by colloidal electrolytes and other compounds was initiated by E. K. Rideal at Cambridge University and was subsequently carried forward by J. H. Sehulman and his collaborators. A portion of their work dealt with the influence of the ionic group of the colloidal electrolyte. More recently they have studied the hemolytic reaction in terms of monolayer processes in- volving cholesterol and other lipids. Suggestive as this work has been, there are still many questions connected with the subject that it makes no claim to elucidate. The present study also includes the effect of molecular structure on hemolytic activity. By extending the range of conditions, it provides a wider foundation upon which to base postulates of mechanism.

Two homologous series of colloidal electrolytes, one anionic and the other cationic, have been used as hemolytic agents a t different temperatures and also in the presence of added n-propanol at two different temperatures. The hemolytic effect of colloidal electrolytes is therefore recorded under the influence of four independent conditions, namely, ionic charge, size of

i Presented before the Division of Colloid Chemistry at the 125th National Meet- ing of the American Chemical Society, Kansas City, Missouri, March 24-April 1, 1954.

2 This paper is based on a portion of a thesis submitted, by A. M. Silverstein, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, to the Facul ty of Rensselaer Polytechnic Inst i tute .

Present address, Division of Laboratories and Research, New York State De- par tment of Health, Albany, N.Y.

157

158 SYDNEY ROSS AND ARTHUR M. SILVERSTEIN

atkyl group, temperature, and the presence of n-propanol. The data can then be systematically examined as a series of comparisons in which three conditions are constant and one is varied, or twelve different comparisons.

MATERIALS AND EXPERIMENTAL TECHNICS

1. Materials

The series of alkyldimethylbenzylammonium chlorides has been used previously in a study of their bactericidal effects and the method of synthe- sis and analytical data are already published (1). These materials were supplied by courtesy of the Sterling-Winthrop Research Laboratories. The trimethyldodecylammonium chloride is a sample generously provided by Dr. H. J. Harwood of the Armour Research Foundation. The pertinent information about this material has also been published previously (2).

The series of sodium alkyl sulfates (3) was kindly provided by Dr. Fred Karush of Children's Hospital, Philadelphia, Pennsylvania, with the exception of the 14-carbon member, which was kindly supplied by Mr. E. Barthel, Jackson Laboratory, E. I. du Pont de Nemours and Company. Dodeeyltrimethylammonium bromide was also provided by Mr. Barthel.

All reagents employed in this study were made up and diluted in 0.05 M phosphate buffer at pH 7.2 made isotonic with sodium chloride.

The red cell suspension used is prepared as follows: sheep blood is drawn into citrate-glucose solution (4) to prevent clotting, stored at 3°-6°C., and used for no longer than one week. Each day a portion of the blood is cen- trifuged and the cells washed three times in isotonic salt solution by decantation. The washed, packed cells are then suspended in buffer to yield a 2.5 %-by-volume suspension, and the suspension is then standard- ized exactly for hemoglobin content in a Coleman Junior Spectrophotom- eter at 545 m~.

Ketene is prepared according to previously described methods (5), and bubbled through a chilled suspension of 5 % sheep cells in isotoIiic NaHCO~ solution. The bicarbonate is used in order to neutralize acetic acid formed during the aeetylation of the primary amino groups of the protein moiety. After 15 minutes of treatment, the cells are washed three times in buffer by decantation and then adjusted to yield the standard 2.5 % suspension. These acetylated cells are used in place of the normal, untreated cells for certain experiments.

2. Hemolysis Technics

The minimum concentration of each detergent necessary to hemolyze 50% of the standard cell suspension is determined as follows. Various dilu- tions of th e detergent in isotonic buffer are prepared and brought to the re- quired temperature. One-tenth of a milliliter of each dilution plus 0.2 ml. of buffer at the same temperature are pipetted into 10 X 75-ram. test tubes,

HEMOLYSIS BY COLLOIDAL ELECTROLYTES 159

and then 0.2 ml. of the s t anda rd red cell suspension is added. The mix ture is allowed to react for 15 minu te s in the water b a t h with shaking. At the end of this t ime, 1.0 mt. of cold 0.15 M NaC1 solut ion is added and the mix ture shaken and immedia t e ly centr i fuged for 3 minu tes to remove all unhemolyzed cells. The degree of hemolysis is then de te rmined by reading in the speet rophotometer , which has been previously s tandardized to read

direct ly the per cent hemolysis wi th the cell suspension used. T h a t concen-

t r a t ion of hemolyt ic agent jus t causing 50 % hemolysis in the 15-minute

in te rva l is called the m i n i m u m hemolyt ic dose (C, s0).

EXPERIMENTAL RESULTS

The de t e rmina t ion of hemolyt ic effectiveness for each m e m b e r of each homologous series was done a t each of two tempera tures , 20.0°C. and 37.0°C., and then each de t e rmina t ion repeated in the presence of 0.50 M n-propanol . I n addi t ion , for fur ther comparison, the sample of t r ime thy l - d o d e c y l a m m o n i u m chloride was inves t iga ted unde r all the same condit ions. The da t a are reported in Tables IA and IB and graphical ly in Figs. 1 and 2

as a series of twelve comparisons.

TABLE IA

The Hemolytic Activities (CBbo) in Moles per Liter of a Homologous Series of Alkyldi- methylbenzyl Ammonium Chlorides in the Presence and Absence

of 0.5 M n-Propanol at 20°C. and 37°C.

Series No propanol 0.5 M n-Propanol member

20°C. 37°C. 20°C. 37°C.

C3 (]1o C12 Cl4 C16 Cls

4.0 X 10-2 3.5 X 10 -~ 2.3 X 10 -4 2.9 X 10 -5 1.3 X 10 -5 2.7 X 10 -5

1.7 X 10 -~ 1.1 X 10 -3 1.5 X 10 -4 3.9 X 10 -5 1.6 X 10 -5 3.0 X 10 -5

2,0 X 10-: 1.3 X 10 -3 1.3 X 10 -4 3.4 X 10 -5 1.4 X 10 -5 3.0 X 10 -5

3.0 X 10 -~ 3.5 X 10 -4 4.5 X 10 -5 2.3 X 10 -5 1.3 X 10 -5 2.5 X 10 -5

TABLE IB

The Hemol. flic Activities (CHbo) in Moles per Liter of a Homologous Series of Sodium Alkyl Su Pates in the Presence and Absence of 0.5 M n-Propanol at 20°C. and 37°C.

Series No propanol 0.5 M ~-Propanol member

20°C. 37°C. 20°C. 37°C.

C8 C1o C1: C14 C16 Cls

1.8 X 10 -2 9.7 X 10 -4 5.9 X 10 -5 2.7 X 10 _5 3.2 X 10 -5 6.3 X 10 -5

1.6 X 10-: 1.1 X 10 -s 9.8 X 10 -~ 3.4 X 10 -5 3.4 X 10 -~ 2.5 X 10 -5

1.7 X 10-2 1.1 X 10 -3 6.1 X 10 -5 3.7 X 10 -5 4.7 X 10 -5 6.8 X 10 -5

3.5 X 10 _3 3.3 X 10 -4 3.8 X 10:5 3.4 X 10 -5 2.2 X 10 -5 2.5 X 10 -5

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H E M O L Y S I S BY C O L L O I D A L E L E C T R O L Y T E S 161

The determination of the hemolytic activity of dodecyltrimethyl- ammonium bromide was done under the same conditions as are listed above and is compared with the results for the C12 members of the two series in Table II.

D I S C U S S I O N

In Figures 1A and IB the effect of temperature is shown on the hemolytic activities of the cationic and anionic detergents, respectively. The cationic series has a positive temperature coefficient for the lower members, be- coming zero at the 14-carbon member and above. The anionic series has zero temperature coefficient throughout, save for the 18-carbon member, which exception may be attributed to the greater solubility of this relatively insoluble material at the higher temperature. Figure 1B agrees with the results of Ponder (6) that the.activity of alkyl sulfates at 20°C. increases with ehainlength and reaches a maximum at C14. The maximum for the quaternary ammonium salts is at C16.

The effect of the presence of 0.50 M propanol is to emphasize the temperature dependence of the hemolytic activity (Figs. 1C and 1D), especially notable for the alkyl sulfates, which previously had little or none. Com- parisons of the effect of propanol at a single temperature (Figs. 2A, 2B, 2C, and 2D), shows that generally the propanol activates hemolysis by members of both series, the smaller chains more than the larger chains. The alkyl sulfates at 20°C., however, are not affected by the presence of 0.50 M propanol.

The remaining diagrams compare the two detergent series directly under the various conditions (Figs. 1E,1F, 2E, and 2F). The alkyl sulfates are less soluble than the corresponding homolog of the quaternary ammonium series, and the higher members of the alkyl sulfate series are not sufficiently soluble to demonstrate their full hemolytic capacity. This phenomenon is akin to the "Ferguson principle," in which the cut-off in activity is ascribed to a solubility effect (7). It is generally evident that the higher alkyl sulfates appear less active than the corresponding quaternary ammonium salts,

T A B L E I I

Comparison of Hemolytic Activities (Cn~o) in Moles per Liter of Dodeeyl-Substituted Colloidal Electrolytes

CI2H2~

- - S O 4 - - - C 6 H s C H 2 - - N +

(CH~)~ - - N + ( C H ~ ) 3

No propanol

20°C.

5 . 9 X 10 -~ 2 . 3 X 10 -4

1 . 2 X 10 - s

37°C.

9 . 8 X 10 -5

1 . 5 X 10 -~

1 . 2 X 10 -3

0.5 M n-Propanol

20°C.

6 . 1 X 10 -~ 1 . 3 X 10 -4

1 . 5 X 10 -3

37°C.

3 . 8 X 10 -~ 4 . 5 X 10 5

3 . 6 X 10 -4

162 SYDNEY ROSS AND A R T H U R M. S I L V E R S T E I N

even though in general the alkyl sulfates are the more active ~hemolytic compounds. Schulman and Armstrong (8) have compared the hemolytic activities of a number of 12-carbon chain detergents and found the alkyl sulfate more active than trimethylammonium salt. We have also observed the same general result (Table II). The order of hemolytic activity

H(CH2)~2--SO( > --C6H~CH2N+(CH~)2 > --N+(CH~)~

is preserved throughout all the various conditions employed in this study. These authors attribute the order of biological activity in part to a stereo- chemical or orientation effect of the ionic group, which can produce con- siderable differences in associating power between two ions or an ion and an induced dipole.

In a recent paper Pethica and Schulman (9) propose the view that hemolysis by saponin and ionic detergents is caused by interaction with the cholesterol portion of the red cell surface. This hypothesis emphasizes the interaction between the lipid portion of the cell membrane and the hydrophobic part of the detergent molecule. The results shown here and other well-known evidence for the increase of hemolytic activity with increasing size of alkyl group, show how important this interaction is. Similar results for an increase of bactericidal action of quaternary ammonium salts with increasing size of the alkyl group have also been interpreted as due to increasing hydrophobic interaction (1).

These considerations, however, need not deny the importance of the concomitant interionic attraction and the significance of the protein portion of the cell surface. It is well known that the protein molecule contains both positive and negative ionic groups. Interaction between proteins and detergents is also well known (10, 11) and has been reviewed by Valko (12) and by Putnam (13). An experimental fact that shows immediately the importance of the ionic group is the comparatively weak hemolytic action of nonionie detergents.

We have, moreover, observed that subhemolytic concentrations of sodium octyl sulfate will inhibit the hemolytic action of sodium hexadecyl sulfate. Thus 10 -2 M sodium octyl sulfate produces a visible reduction in the rate of hemolysis by sodium hexadeeyl sulfate, and a corresponding reduction in its hemolytic potency. This competition between the 8- and 16-carbon atom anions on the red cell surface is similar to that observed by Karush (14) between an anionic azo dye and sodium dodecyl sulfate on bovine serum albumin. The similarity is suggestive, since Karush interprets his results as competitive interaction between the organic anions for the oppositely charged sites on the protein molecule.

Perhaps the most direct evidence of the importance of the ionic groups on the erythrocyte surface as the primary site of hemolytic attack by ionic detergents is the effect of the acetylation of the amino groups on the erythro-

H E M O L Y S I S B Y C O L L O I D A L E L E C T R O L Y T E S 163

cyte surface by ketene. After thus reducing the number of surface cationic groups, the hemolytic activity of an anionic detergent is considerably lowered, while that of a cationic detergent is practically unimpaired. (These experiments are reported here for the first time.)

The connection of the biological action of quaternary ammonium salts (same salts as discussed in this paper) and their colloidal properties has been discussed by Ross, Kwartler, and Bailey (1). The increase of effectiveness with the number of carbon atoms is shown both in the hemolytic action and in the antibacterial action of these quaternary ammonium salts. The maximum action occurs at Cls for hemolysis and at C14 for the bactericidal effect. The presence of the maximum may be accounted for in the same way in both cases, namely, as a competition for detergent molecules between the cell surface and the colloidal micelle. Since micelle formation increases more rapidly than biological activity, as a function of the number of carbon atoms, the higher members of the series begin to show a declining hemolytic and bactericidal effect. Both in the hemolytic and in the bactericidal measurements, the concentrations used are in the same range as the critical micelle concentrations for C16 and C18-alkyl groups, and it is precisely here that the effect of micelle formation influences the biological effectiveness. Low solubility of the detergents also influences biological effectiveness in the same way as does pronounced micelle formation and, for the alkyl sulfates, both low solubility (for the C~s member at 20°C.) a~ld increased micelle formation contribute to produce the maximum.

For the lower member chains, micelles in the bulk solution should not affect the hemolytic action, as the critical micelle concentration is far greater than the concentrations at which hemolysis occurs. It is therefore not to be expected that any relation can be found between CMC and hemolytic action for these lower members. Flockhart and Ubbelohde (15) have measured, by electrical conductance, the CMC of sodium lauryl sulfate in water and in water-propanol mixtures. Examination of their results reveals that although the presence of propanol and differences in temperature cause relatively large variations in CMC, these variations are not paralleled by the variations of the hemolytic action. This is shown by the results reported in Table III.

Variation of temperature and the presence of n-propanol affect the hemolytic activity of detergents containing low-member chains in a manner that cannot be described as caused by their effect on micelle formation. It is only when the CMC is low, indicating a strong tendency to form micelles, that micelle formation can clearly be discerned as directly in- fluencing the biological activity. Indirectly, the CMC may be taken as a measurement of surface activity, and the lack of correlation shown in Table III suggests mechanisms of hemolysis not related to the surface effects stressed by Pethica and Schulman.

164 S Y D N E Y ROSS AND A R T H U R M. S I L V E R S T E I N

TABLE I I I

Comparison of CMC ~ and Hemolytic Activity of Sodium Dodecyl Sulfate in the Presence and Absence of 0.5 M n-Propanol at 20°C, and 37°C.

No propanol 0.5 M n-Propanol Moles/liter

20°C. 37°C. 20°C. 37°C.

CHso 5.9 X 10 5 9.8 X 10 ~5 6.1 X 10 -5 3.8 X 10 -5 CMC ~ 8.2 X 10 -~ 8.0 X 10 -3 5.3 X 10 -3 5.6 X 10 -3

a The values for CMC are interpolated from Fig. 6 of the paper by Floekhart and Ubbelohde (15).

I t has been pointed out by Love (16) that sodium dodecyl sulfate has unusual hemolytic action on human erythrocytes. A short initial period of rapid hemolysis is followed by an interruption of cell destruction that may last for many minutes before, finally, the disappearance of the remaining Cells. This behavior is quite unlike the usual sigmoid curves found for kinetic hemolysis by complement, by butyl alcohol, or by distilled water, which show a period of lag before the onset of hemolysis. The following remarks by Love (16) have an important bearing on the present work.

"The relatively simple hemolytic action o f . . . exposure to butyl alcohol is considerably modified in the case of sodium dodecyl sulfate, sodium oleate, and sodium taurocholate, all of which show an anti-hemolytic as well as hemolytic action. I t is perhaps significant that the three compounds which have so far been found to have this additional effect are those which have a marked capacity for forming micelles. Their protective action may be a result of a tendency to aggregate with neighboring molecules when combined in large numbers with the cell surface."

During the course of the present work, hemolytic effects similar to those described by Love were observed. The magnitude of the instantaneous initial hemolysis is most pronounced with the higher members of the homologous series, both quaternary ammonium salts and alkyl sulfates, and is progressively less pronounced for the smaller alkyl groups. Below dodeeyl the effect is imperceptible. This observation may be expected if Love's suggestion is true, namely that the unusual hemolytic action is characteristic of substances with a marked capacity for forming micelles. The tendency to form micelles decreases exponentially with a decrease in the size of the alkyl group (1).

I t was also observed in the course of this work that the presence of small amounts of n-propanol down to 0.10 M, causes the disappearance of the unusual hemolytic effect obtained from the higher members of both series. The presence of such low concentrations of n-propanol affects the bulk CMC of the detergents only slightly (cf. Table III) , but effects tha t may occur at the erythrocyte surface are not necessarily described by

HEMOLYSIS BY COLLOIDAL ELECTROLYTES 165

observations of bulk phenomena of detergent solutions in the absence of erythrocytes. The dielectric constant at the surface may be altered by n-propanol and surface aggregation greatly reduced as a consequence.

ACKNOWLEDGMENTS

A portion of the experimental work described in this paper was done in the laboratories of the Division of Laboratories and Research, New York State Department of Health, Albany, New York. The authors acknowledge the assistance of Dr. F. C. Nachod, Sterling-Winthrop Research Laboratories, who provoked a thorough dis- cussion of the contents of the paper.

]~EFERENCES

1. Ross, S., KWARTLER, C. E., AND BAILEY, J. H., J. ColloidSci. 8, 385 (1953). 2. CELLA, J. A., EGGENBERGER, D. N., NOEL, D. R., HARRIMAN, L. A., .4_ND HAR-

WOOD, H. J., J. Am. Chem. Soc. 74, 2061 (1952). 3. KARUSH, F., AND SONENBERG, M., g. AFt. Chem. Soc. 71, 1369 (1949). 4. BUKANTZ, S. C., REIN, C. R., AND KENT, J. F., J. Lab. Clin. Med. 31,394 (1946). 5. H~RRrOTT, R. M., J. Gen. Physiol. 18, 69 (1934). 6. PONDER, E., J. Gen. Physiol. 30, 15 (1946). 7. FERGUSON, J., Proc. Roy. Soc. (London) 127B, 387 (1939). 8. SCHUL~IAN, J. H., AND ARMSTRONG, W. McD., "Surface Chemistry," pp. 273-279.

Interscience Publishers, Inc., New York, 1949. 9. PETHICA, B. A., AND SCHULMAN, J. H., Biochem. J. 53, 177 (1953).

10. PUTNAM, F. W., AND NEURATH, H. J., J. Am. Chem. Soc. 66: 692, 1992 (1944). 11. SCI-IMIDT, K. H., Z. physiol. Chem. 277, 117 (1942). 12. VALKO, E. I., Ann. N. Y. Aead. Sci. 46, 451 (1946). 13. PUTNAM, ]7. W., "The Interactions of Proteins and Synthetic Detergents," (152

references). Advances in Protein Chem. 4, 79-122, (1948). 14. KARUS~, F., J. Ant. Chem. Soc. 72, 2714 (1950). 15. FLOCKHART, B. D., AND U]~BELOHDE , A. R., J. Colloid Sci. 8, 428 (1953). 16. LOVE, L. H., J. Cellular Comp. Physiol. 36, 133 (1950).

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Hemolysis by colloidal electrolytes