Interaction of histidine analogs with zinc phosphate dispersion

Interaction of Histidine Analogs with Zinc Phosphate Dispersion and the mixtures were titrated with 0.2 N NaOH to pH 7.0. Precipitation of amorphous zinc phosphate took place at about p i t 4.0 and was completed at about p t I 6.2. The volume was made up to 50 ml with freshly distilled water; then sodium chloride was added to adjust the ionic strength of the system to 0.1. The sample was divided into two portions; stabilization was examined on one part, histidylhistidine binding on the other. The amount of histidylhistidine bound to zinc phosphate was determined by measuring unbound histidylhisfidine in the supernatant solution according to Macpherson (3) after 10 rain centrifugation at 3000 rpm. 1- and 3-methylhistidine, which do not develop color with sulfanilic acid, were determined by ninhydrin. At the same time, the concentration of zinc and phosphate in the supernatant were analyzed by dithizone and rnolybdate, respectively, to check that the amount of zinc phosphate precipitated was unaltered by the addition of histidylhistidine. When the amount of bound histidylhistidine (r) was plotted against unbound histidylhistidine ( C f ) , a parabolic curve was obtained (Fig. 1). Apparent binding constant k and maximum binding n were calculated by the Klotz equation (4) with the results of k = 1.7 X 104 and n = 1.9. By a series of experiments, the apparent binding constants of histidine analogs were calculated (Table I) but calculation of the constants for N-acetylhistidine or 3-methylhisfidine was not possible because of their very poor affinity for zinc phosphate. As shown in the table, histidinol and 1-methylhistidine were found to have the same affinity as histidine, whereas histidine methyl ester and imidazole lactic acid have

Many reports have appeared dealing with zinchistidine interactions, much attention being paid to the nature of the coordinated ligand (1) and to the structure-activity relationship of biological macromolecules (2). In these, an important role of the imidazole moiety of histidine in the interaction is emphasized. We have examined the interaction in a zinc phosphate dispersion system and found that stereospecific coordination is involved in the heterogeneous system. When an acidic zinc phosphate solution was neutralized, a flocculent amorphous precipitate was formed which aggregated during 24-hr storage at 25°C. If, however, hisfidine was included in the system, the aggregation was prevented and the flocculent dispersion was maintained for several months. We then examined whether zinc phosphate dispersion can similarly be stabilized by interaction with histidine analogs, and found that several analogs serve as the stabilizers. A dispersion containing 3 mM histidylhistidine still retained its initial sedimentation properties after 1 yr of storage at 45°C. Experiments were performed as follows: 10-ml-samples of zinc phosphate solution (60 mmole zinc chloride in 1000 ml of 40 mM phosphoric acid) were mixed with 25 ml of histidylhistidine solution of varying concentration (2-10 mM),

1.5

TABLE I

1.0

]3INNING PARAMETERS OF HISTIDINE ANALOGS

[r] n

L-histidyl-L-histidine 5-histidyl-L-alanine fl-alanyl-L-histidine L-histidine nL-histidine Jrl-methylhistidine Histidinol ~-histidine methyl ester L-fl-imidazole lactic acid

0.5 Io0

f

0.1 [Cf]

2OO

I

0.2 m'l~

1.9 0.5 0.6 0.9 0.9 0.7 1.0 0.8 0.7

k (L/M)

1.7 X 4.3 M 2.5 X 3.6 X 3.6 N 3.7 M 4.2 X 1.2 X 1.4 X

104 102 102 102 102 102 102 102 102

Fro. 1. Binding of histidylhisfidine by zinc phosphate. 352 Journal of Colloid and Interface Science, Vol. 48, No. 2, August 1974

Copyright ~ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

NOTES significantly low affinities. These results suggest that imidazolyl nitrogen at position 3 and carbonyl residue participate in the interaction. As expected, nonimidazolyl amino acids such as glycine, tryptophane, and phenylalanine did not bind zinc phosphate to any appreciable extent. Acetylation of the a-amino residue completely abolished the affinity, probably by interfering with the access of the histidine molecule to zinc phosphate. The lower affinity of histidylalanine and /3-alanylhistidine may be due to steric hindrance. As stated above, several hisfidine analogs rendered zinc phosphate stable to storage. The extent of the stabilization depended cn the structure of the analogs; histidine methyl ester, imidazole lactic acid and histidylalanine were less effective than histidine; on the other hand, histidylhistidine was highly effective. Histidinol and 1-methylhistidine were as effective as histidine; but n-acetylhistidine and 3-methylhistidine, like glycine, tryptophane, and phenylalanine, were almost ineffective under the condition employed. Thus, the stabilizing ability and binding affinity of histidine analogs are closely related. An interesting finding was stereoselectivity in stabilization: zinc phosphate with 5 mM DL-histidine aggregated in 20 days at 25°C, but that with L- or n-histidine did not change over several months, though the apparent binding constants of the 3 enantiomers are essentially the same. A similar result was obtained when an equivalent amount of the zinc

353

phosphate with L- or D-histidine was combined, suggesting an interaction between two forms of the particles. Preliminary electron microscopic studies revealed that the dispersion is composed of spherical particles, approximately 30 nm in diameter, which seem to be unaltered by the presence or absence of histidine. Bound histidine may alter the hydrophobic surface nature of the primary particle to the hydrophilic one, thus preventing coagulation of the dispersion during the storage. REFERENCES 1. HARDING, M. H., AND COLE, S. J., Acta Cryst. 16, 643 (1963). 2. BRILL, A. S., AND VENABLE, ]. H., J. Amer. Chem. Soe. 89, 3622 (1967). 3. MACPKERSON, H. T., Biochem. J. 36, 59 (1942). 4. KLOTz, I. M., "The Proteins," Vol. I, p. 727. Academic Press, New York, 1953. MASAI~ARU HIRATA TAKAKO TAMURA MASAYA BABA

Shionogi Research Laboratory Shionogl 6~ Co., Ltd. Fukushima-ku, Osaka, 553 Japan Received December 27, 1973; accepted February 12, 1974

Journal of Colloid and Interface Science. Vol. 48. No. 2. August 1974