Biochirnica et Biophysica Acta, 427 (1976) 337-349
© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37254 T H E P A R A D O X I C A L E F F E C T OF F A T T Y A C I D O N S T E R O I D - A L B U M I N INTERACTION
MICHAEL T. RYAN* and RAVI K. CHOPRA Department of Biochemistry, Faculties of Medicine and Science, University of Ottawa, Ottawa, Ontario KIN 9A9 (Canada)
(Received June 24th, 1975)
SUMMARY Careful investigation of the influence of palmitic and lauric acid on the interaction of progesterone and testosterone with several batches of untreated and defatted bovine and human serum albumins have revealed that, by contrast with published data for studies with progesterone as well as nonsteroid ligands, there is a surprising stimulation rather than inhibition of binding, albeit with a reduction of the apparent number of binding sites in almost all instances. Furthermore, fatty acid tends to minimize or eliminate the well-known differences in affinity between bovine and human albumin for interactions with these two steroids. The values for binding affinity in the interaction of testosterone with these batches of human serum albumin are significantly higher than those previously published by us and other authors and the value for progesterone-bovine albumin interaction is not in accordance with the "polarity rule". Studies of these same interactions by ultraviolet difference spectroscopy give further evidence of the augmentation in binding but, in the case of defatted bovine albumin only, the aromatic difference troughs are indicative of tyrosine perturbation whereas refatted bovine albumin, defatted and refatted human albumin manifest tryptophan perturbation. Quantitative correlation of perturbation with level of bound steroid suggests that fatty acid alters the ratio (possibly hydrogen-bonded to non hydrogen-bonded) of two forms of bound steroid. There is also further evidence that the binding sites for testosterone and progesterone are not identical.
Serum albumin has long been known for its ability to bind a large variety of organic and inorganic ligands. The organic compounds include such physiological substances as thyroxine, tryptophan [1] bilirubin [2] steroid hormones [3] and such nonphysiological substances as detergents, long-chain alcohols [1] and drugs [4]**. In the latter case the phenomenon may have considerable pharmacological significance. We have undertaken a systematic study of various facets of steroid-albumin interaction [5-8], in the course of which we have turned to a study of the influence of * To whom requests for information should be addressed. ** A list of papers dealing with fatty acid inhibition of binding of other ligands may be found in this paper.
338 fatty acid on the phenomenon. Fatty acids are bound to a total of 8-9 sites [9] while testosterone is bound to a minimum of two sites [3], but higher values have been reported [3, 7]. Ultraviolet difference spectroscopy has been used extensively in studying the state of aromatic amino acids and other structural features in proteins and of alterations induced by conformational change and ligand binding [10]. Thus, for example, the interaction of fatty acid and human albumin produces a characteristic red-shifted difference spectrum [11, 12] identical in the location of its troughs to one produced by 3 [/3]-hydroxy-androst-5-en-17-one, similar to one produced by testosterone, cortisol, progesterone, [6] and many long-chain hydrocarbon ligands [13]. In addition, the latter three steroids exhibit a difference peak at 259 nm and the spectral effects are explainable in terms of hydrogen bonding of the a, fl-unsaturated steroid carbonyl to a tryptophan residue located at a hydrophobic binding site [6]. It is thus not surprising that, in keeping with the results of those for other ligands (eg ref. 4), studies with defatted and refatted protein have indicated an apparent noncompetitive inhibition of binding of progesterone to human albumin by myristic, lauric and palmitic acids [14, 15]. However, we now report data for the binding of progesterone and testosterone to untreated, defatted, deionized bovine and human albumin and to albumin preparations which have been refatted with 3 mol palmitic acid per mol protein and in which stimulation of binding by fatty acid is seen. There are 8 heterogenous sites in human albumin for palmitic acid [9]; the single high-affinity site (K~ss, 3.2.10 7) and the succeeding two of comparable K, ss (7.79.106) would be essentially filled by 3 mol palmitic acid, since the next site has a/
© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37254 T H E P A R A D O X I C A L E F F E C T OF F A T T Y A C I D O N S T E R O I D - A L B U M I N INTERACTION
MICHAEL T. RYAN* and RAVI K. CHOPRA Department of Biochemistry, Faculties of Medicine and Science, University of Ottawa, Ottawa, Ontario KIN 9A9 (Canada)
(Received June 24th, 1975)
SUMMARY Careful investigation of the influence of palmitic and lauric acid on the interaction of progesterone and testosterone with several batches of untreated and defatted bovine and human serum albumins have revealed that, by contrast with published data for studies with progesterone as well as nonsteroid ligands, there is a surprising stimulation rather than inhibition of binding, albeit with a reduction of the apparent number of binding sites in almost all instances. Furthermore, fatty acid tends to minimize or eliminate the well-known differences in affinity between bovine and human albumin for interactions with these two steroids. The values for binding affinity in the interaction of testosterone with these batches of human serum albumin are significantly higher than those previously published by us and other authors and the value for progesterone-bovine albumin interaction is not in accordance with the "polarity rule". Studies of these same interactions by ultraviolet difference spectroscopy give further evidence of the augmentation in binding but, in the case of defatted bovine albumin only, the aromatic difference troughs are indicative of tyrosine perturbation whereas refatted bovine albumin, defatted and refatted human albumin manifest tryptophan perturbation. Quantitative correlation of perturbation with level of bound steroid suggests that fatty acid alters the ratio (possibly hydrogen-bonded to non hydrogen-bonded) of two forms of bound steroid. There is also further evidence that the binding sites for testosterone and progesterone are not identical.
Serum albumin has long been known for its ability to bind a large variety of organic and inorganic ligands. The organic compounds include such physiological substances as thyroxine, tryptophan [1] bilirubin [2] steroid hormones [3] and such nonphysiological substances as detergents, long-chain alcohols [1] and drugs [4]**. In the latter case the phenomenon may have considerable pharmacological significance. We have undertaken a systematic study of various facets of steroid-albumin interaction [5-8], in the course of which we have turned to a study of the influence of * To whom requests for information should be addressed. ** A list of papers dealing with fatty acid inhibition of binding of other ligands may be found in this paper.
338 fatty acid on the phenomenon. Fatty acids are bound to a total of 8-9 sites [9] while testosterone is bound to a minimum of two sites [3], but higher values have been reported [3, 7]. Ultraviolet difference spectroscopy has been used extensively in studying the state of aromatic amino acids and other structural features in proteins and of alterations induced by conformational change and ligand binding [10]. Thus, for example, the interaction of fatty acid and human albumin produces a characteristic red-shifted difference spectrum [11, 12] identical in the location of its troughs to one produced by 3 [/3]-hydroxy-androst-5-en-17-one, similar to one produced by testosterone, cortisol, progesterone, [6] and many long-chain hydrocarbon ligands [13]. In addition, the latter three steroids exhibit a difference peak at 259 nm and the spectral effects are explainable in terms of hydrogen bonding of the a, fl-unsaturated steroid carbonyl to a tryptophan residue located at a hydrophobic binding site [6]. It is thus not surprising that, in keeping with the results of those for other ligands (eg ref. 4), studies with defatted and refatted protein have indicated an apparent noncompetitive inhibition of binding of progesterone to human albumin by myristic, lauric and palmitic acids [14, 15]. However, we now report data for the binding of progesterone and testosterone to untreated, defatted, deionized bovine and human albumin and to albumin preparations which have been refatted with 3 mol palmitic acid per mol protein and in which stimulation of binding by fatty acid is seen. There are 8 heterogenous sites in human albumin for palmitic acid [9]; the single high-affinity site (K~ss, 3.2.10 7) and the succeeding two of comparable K, ss (7.79.106) would be essentially filled by 3 mol palmitic acid, since the next site has a/
