Letter pubs.acs.org/JPCL
Reversible Insulin Hexamer Assembly Promoted by Ethyl Violet: pHControlled Uptake and Release Jyotirmayee Mohanty,*,†,# Meenakshi N. Shinde,†,‡ Nilotpal Barooah,† and Achikanath C. Bhasikuttan*,†,# †
Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400 094, India ‡ Student, BARC-SPPU PhD Program, Department of Chemistry, Savitribai Phule Pune University, Pune 411 007, India #
S Supporting Information *
ABSTRACT: Therapeutically improved long-acting insulin preparations require in-depth understanding of the hexamer assembly, structural selectivity, and its stability in solution. This Letter demonstrates, for the first time, an efficient method for the hexamerization of human insulin by a structure-specific triphenylmethane (TPM) dye, Ethyl Violet (EV), particularly, in the absence of Zn2+. Upon detailed spectroscopic evaluation and comparison with other TPM homologues, we establish that the diethylamino phenyl arms of EV are specific and effective in clipping the three dimer helices in a hexameric assembly. We establish that at physiological pH 7.4 and in the presence of the EV, insulin exists predominantly in its hexameric form, a condition appropriate for storage and preparation of long-acting insulin formulations. On the other hand, the disassembly of the hexamer into the monomeric form is accomplished at pH 5, highlighting its potential as a delivery vehicle for such custom-modified dyes/drugs.
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stored in the pancreatic vesicles in a hexameric form (Scheme 1), complexed with zinc ions, and is released in response to the
ransitions to structure-specific self-assemblies are quite prevalent in the functional pathways of several biomolecules, prominently in proteins and DNAs.1−3 The dynamic assembly of these bioentities into receptor binding templates or storage modules is usually stabilized by metal ions, hydrogen bonds, dispersion forces, and the entropic benefits associated with the consequential structural changes.4,5 Often these transformations are under significant influence by ambient conditions like pH, ionic strength, temperature, and smallmolecule binding. In this context, insulin, a small helical protein hormone, has been extensively discussed for its structural/ functional details and its physiological role in regulating blood glucose levels, its biochemical synthesis and pharmacological properties, in the treatment of diabetes and its recombinant pharmaceutical preparations.6,7 Insulin consists of two polypeptide chains, the A chain having 21 amino acid residues and the B chain having 30 amino acid residues, linked together by two interchain disulfide bridges. One of its most striking characteristics is its ability to form different association states including dimers, tetramers, and hexamers.8−10 Moreover, under denaturing conditions, such as low pH or in the presence of strong denaturants, insulin aggregates into amyloid fibrils, which are believed to cause cellular dysfunction responsible for Alzheimer’s and Parkinson’s diseases and so forth and has been the subject of extensive studies by us11,12 and several other groups.13,14 Understanding the structure of insulin is primarily motivated by the need to produce modified long-acting insulins for use in the treatment of diabetes.15 Though the metabolic action of insulin in vivo is initiated by the binding of monomeric insulin to the specific receptors on the cell membrane, the protein is © 2016 American Chemical Society
Scheme 1. Chemical Structures of the TPM Dyes and the Structure of the Insulin Hexamer
blood glucose level.16 The hexameric moiety is relatively resistant to degradation/fibrillation, and it is the zinc hexamer that is the primary component of all insulin pharmaceutical preparations, which allows for a more constant basal level of insulin in diabetics.6,17−19 Modification of any factors, intrinsic (by mutations20) or extrinsic (by metal ions or other additives4,5,21), that influence the self-association and dissociation of insulin or its aggregates is crucial in the production of Received: August 5, 2016 Accepted: September 23, 2016 Published: September 23, 2016 3978
DOI: 10.1021/acs.jpclett.6b01745 J. Phys. Chem. Lett. 2016, 7, 3978−3983
Letter
The Journal of Physical Chemistry Letters long-acting insulin formulations so as to optimize the bioavailability during therapeutic action.21 In the recent past, several studies have been reported analyzing the potential of different types of species such as phenolic moieties in stabilizing the hexameric structures prepared in the presence of Zn2+.9,19,20,22 Phenolic additives bind to specific sites on the insulin hexamer and act as allosteric effectors, inducing a transformation among different hexamer structures by providing specific interactions at the dimer−dimer interface that stabilize the B1−B8R helices.9,19 Studies have also been carried out on the inhibition of islet amyloid polypeptide (IAPP) aggregation by the hexameric state of insulin, which in turn is regulated by Zn2+ concentration.23 On the other hand, small-molecule binding to macrobiomolecules proved effective in influencing the biofunctionalities through their cooperative effect.24,25 In this context, triphenylmethane (TPM) dyes have gained much attention due to their aptness to cooperatively bind to biomolecules, which finds relevance in many phototherapeutic and sensor applications.26−30 The radiative lifetimes of TPM dyes are very short (
