September 1969
1IOL)EL FOR ~ ~ S T 1 H l Y T A A l I I U lACC T I O N
This technique permits us to exainiiie the molecular events which occur during histamine binding to bovine serum albumin. I n several antihistamiriics of different chemical composition the antihistaminic activity ran parallel to the effect of the ionic strengthjc on the structural stability of bovine serum albumin. It has been previously demonstratedjcre that the bovine serum albumiIi-histamine interaction is dependent on ionic strength, that is, there is a critical saline coricentration which can be regarded as a threshold defining two sharply different behaviors of bovine serum albumin in the presence of histamine. It was suggested that bovine serum albumin undergoes structural changes which seem to be triggered by histamine. These structural changes are regulated by the ionic strength of the medium which hinders the attachment of histamine to the macromolecule when a critical salt coiicentration is reached. Since a n increase in the total ionic strength of the medium will reduce the electrostatic interaction between opposite charges,6 this limiting influence could riot account for either the structural change or the threshold significance of the effect.
Results and Discussion Specific conductivity measurements of histamine solutions a t different concentrations have shown that 110 aggregation takes place in the range of concentration used in these studies. At the same time no discontinuity can be detected by specific conductivity of a bovine serum albumin solution (in S a C l 1 X lov4J I ) of increasing concentration. In Figure 1, specific conductivity, x, is plotted a;, u furictiori of histamine molarity in a solution of bovinc serum albumin whose concentration is kept constarit (1 mg/ml) and contains differing amounts of antihistaminics. g
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Figure 1 .--Specific ccindrictivity, x, us. hibtamiire molai.ily i l l a solution of boviiie serum albumin (1 mg,”l) coiitairiirig dipheiihydramirie hydrochloride ( G O , 1 X 10-251; 0-0, 1 X 10-421f).
standard calomel electrode
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reference saline solution
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bovine 1 serum albumin 1
’ reference 112
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’ standard saline calomel solution I electrode
(6) P. Debye and E. Huckel, Physik. Z., 14, 185 (1923).
1
The plots refer to two different experimental conditioris which are characterized by the concentration, 1X and 1 X -11,respectively, of the antihistaminics present in the medium and whose value is kept unaltered throughout the measurements. The transition point in one of the two plots of this figure marks the “complex” formation and gives the value for the ratio by weight of histamine to bovine serum albumin in the complex. At the same time no discontinuity and therefore no binding can be detected in the other plot. Thus an increase in the concentration of the antihistaminic present inhibits the binding of hiitamine to the macroion in the range of concentrations studied. Similar results (Figure 2 ) mere obtained in investigating the bovine serum albumin-histamine-antihistaminics system by means of membrane equilibrium dialysis according to a procedure proposed by Iilotz arid Walther.’ Here the diffusion of histamine occurs at two different rates. When the antihistaminic concen.I1 t h e tration is above the critical value of 1 x iysteni behave, as if the nondiffusible macromolecule w a h not present arid hiitamine diff’uies through the dialysiq membrane accordirig to i t \ concentration gradient. Hy contra\t, when the aiitihistamiiiic concentration is equal to 1 X XI, the hiqtnmiiir i i d d d I * “c:ipt u r d ” Ly the biopolyiiier. Tlierefoie iiieiiibraiie equilibrium dialysis further substantiates the observ~d effects which occur for concentration values of the constituents approximating those found through conduc tivity measure men t s. ( 7 ) J RI Klotz and F. h l . naltlier, J A m Chem Soc 7 0 , 943 (lQ48).
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Pigiire 5.-Specific conductivity, x, us. histamine molarity in a solution of bovine serum albumin (1 mg/ml) containing procaine hydrochloride (1 X M).
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2
3
4
5
6
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Histamine, M X 103
Figlire 7.--Specific conductivity, X, 2)s. histamine molarity i n R solution of bovine serum albrimin i l mgiml) in the preseiice of hydrocortisone-21 sodium succinate at two different molar 31). concent,rations ( G O , 1 X 10-4 .If: 0-0, 1 X I 40
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Figure 6.-Electromotive force (in mV) us. logarithm of histamine concentration in a solution of bovine serum albumin (1 mglml) 31). containing procaine hydrochloride (1 X
serum albumin whoqe concentration is kept constant (1 mg /ml) in the presence of hydrocortisone-21 sodium succinate a t two diff erent values of concentration (1 X lop4 and 1 X 10-5 M). In Figure 8 , the electromotive force (in mV) recorded in a solution of bovine serum albumin (1 mg,”l) containing different amounts (1 X arid 1 X 10-5 M) of hydrocortisone-21 qodium succinate is plotted us. the logarithm of histamine molarity. I n both these cases the electrochemical data indicate that competition exists between cortisol and histamine and that a well-defined concentration of cortisol inhibits the binding of histamine to the macroion. By compiiriiig Figurw 3 arid 1,one finds that t h c value:, o f 1 1 1 ~1worded electroinotivc force and tlic slope of the first part of the plots shoTv that a t this stage the antihistaminics are being “dislocated” from the bovine serum albumin while histamine takes their p1:wi. A ( t l l c Y I I I I ( ~ t i i i i c s (IIV4 i f ‘ i i n pH (frolu -1.7 t o 4.2) contributes to the increase in ionic activity a. indicated by the decrease in the recorded electromotive force. The slope of the second part of the plots s h o w
Mh.,”
Figure 8.-Electromotive force (in mV) us. logarithm of histamine molarity in a solution of bovine serum albumin (1 mg ml) containing diff erent amounts of hydrocortisone-21
