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A specific method for the detection of small quantities of insulin in body tissues and fluids might help to resolve some of the problems connected with the mechanism of action and hormonal interrelationships of insulin. The biological assay methods for small quantities of insulin (2-4) are based upon the effect of insulin on glucose metabolism. Although very sensitive, these methods have several disadvantages. They are technically difficult, are dependent upon the physiological effect of insulin, therefore, subject to considerable biological variation, and consequently may not be specific for insulin per se. A method specific for insulin and independent of biological variation would, therefore, be highly desirable.

Certain relationships of the chain folding within the monomer of bovine insulin in solution have been studied by covalently labeling the three amino groups of insulin with an optical probe. Three purified derivatives containing from one to three groups of the symmetric dye fluorescein isothiocyanate bound to the B-1; B-1 and A-1; B-1, A-1, and B-29 residues , Biochemistry 6, 2378) were examined by absorption and circular dichroism spectroscopy between 400 and 550 nm. The results were interpreted according to the simplified exciton model. This approach was confirmed by the examination of three model compounds prepared by the covalent addition of the dye group to models of the insulin sequence at the sites of substitution and a fourth model compound prepared by disubstitution of L-lysine. These monosubstituted model compounds had absorption spectra similar to the free dye, maxima at 489-492 nm, were nearly optically inactive (400-550 nm), and had similar probe group pK values, average 6.46 i 0.03. In contrast, disubstituted lysine was 5 4 x hypochromic, revealed two new absorption bands at i 866 cm-' about the monosubstituted P revious investigations of the structure-function relationships of insulin by immunochemical, biological and physical techniques Bromer et al., 1967; Mercola et ul., 1967 Mercola et ul., , 1969 Morris et ul., 1968 Morris et ul., -1970a have led to a model of certain chain relationships and conformational features necessary for high immunological and biological Clin. Med. 74, 698. 242,1294. maximum (cu. 20,320 cm-I) with molar circular dichroism values of +15.4 and -18.0 M-' cm-', respectively, had a single pK of 7.28 i 0.06, showed mutarotation, was poorly fluorescent and revealed a maximum probe separation of 7.4 A all in agreement with a dye-dye complex involving exciton formation. The insulin derivatives were shown to be monomeric below 5 X M by comparison of sedimentation (10-2-10-3 hi) and Beer's law behavior (10V to 4 X lo-' hi). Monomeric di-and trisubstituted insulins revealed circular dichroism maxima at about i 6 2 0 and 1 2 3 0 cm-l about the monosubstituted maximum with molar circular dichroism of -10.0, 4-21.5, -32.0, and t 9 1 . 5 , respectively. The results revealed that the B-1 to A-1 and A-1 to B-29 dye group separations were at most about 7.6 and 10.5 A, respectively. These results support certain tertiary relationships previously postulated as necessary for high immunological and biological activity (Arquilla e f al. (1969), Diabetes 18, 193) and it is argued that much of the crystalline structure of rhombohedral two-zinc porcine insulin remains invariant in solution.activity (Arquilla et ul., 1969). In an effort to refine these relationships we have explored a new use of a spectroscopic technique involving exciton formation and have initiated its application to insulin. The stimulus for this approach was the availability of the techniques for the preparation and purification of three insulin derivatives covalently labeled with one of D. A . M. (Uiiivcrsity of Cal...

Experiments were designed to study the effect of Zn on in vivo and in vitro insulin metabolism. The in vivo experiments involved pretreating mice with either Zn or Na, followed by ip [125I]iodoinsulin injection. Pretreatment of mice with Zn resulted in an accelerated and increased magnitude of binding of [125I]iodoinsulin to the liver compared to mice pretreated with Na. Results are submitted which support the probability that the changes in the amounts of intact and degraded insulin in circulation with time are related to the binding and degradation of insulin in the liver rather than in the kidney. In vivo ip injected insulin was demonstrated to preferentially bind to the plasma membrane of the liver. Liver plasma membranes isolated from mice pretreated with Zn bound more [125I]iodoinsulin than plasma membranes of Na-pretreated mice. In vitro experiments employing isolated liver plasma membranes demonstrated that added Zn increased the binding and inhibited the degradation of insulin. Evidence is presented that supports the concept that two receptors exist, one at which degradation of [125I]iodoinsulin occurs and another at which degradation does not occur.

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