Insulin molecular mass

Insulin, a small protein of molecular mass 6000 daltons, is composed of two chains designated A and B. There are no reduced cysteine residues in insulin, but it contains three essential disulfide bonds two that crosslink the A and B chains, and one internal to the A chain to stabilize the overall tertiary stmcture. These disulfide bonds are cleaved in the presence of excess AuX4, leaving A and B chains that have cysteine residues that have become oxidized to sulfonic adds [119]. With smaller amounts of AuX4, a single disulfide bond will be attacked to form sulfinic acid [119]. The reaction is second order for AuCU while AuBr4 reacts too quickly for accurate monitoring. 

Parenteral administration is not perceived as a problem in the context of drugs which are administered infrequently, or as a once-off dose to a patient. However, in the case of products administered frequently/daily (e.g. insulin to diabetics), non-parenteral delivery routes would be preferred. Such routes would be more convenient, less invasive, less painful and generally would achieve better patient compliance. Alternative potential delivery routes include oral, nasal, transmucosal, transdermal or pulmonary routes. Although such routes have proven possible in the context of many drugs, routine administration of biopharmaceuticals by such means has proven to be technically challenging. Obstacles encountered include their high molecular mass, their susceptibility to enzymatic inactivation and their potential to aggregate. 

Pulmonary delivery currently represents the most promising alternative to parenteral delivery systems for biopharmaceuticals. Delivery via the pulmonary route moved from concept to reality in 2006 with the approval of Exubera, an inhalable insulin product (Chapter 11). Although the lung is not particularly permeable to solutes of low molecular mass (e.g. sucrose or urea), macromolecules can be absorbed into the blood via the lungs surprisingly well. In fact, pulmonary... 

Glucagon is a single-chain polypeptide of 29 amino acid residues and a molecular mass of 3500 Da. It is synthesized by the A-cells of the islets of Langerhans, and also by related cells found in the digestive tract. Like insulin, it is synthesized as a high molecular mass from which the mature hormone is releases by selective proteolysis. 

Structurally insulin is a small peptide, with a molecular mass of around 5500 and composed of two subunits, denoted a and (3 chains. Insulin is synthesized as a single peptide, Proinsulin and stored within the pancreatic p-cells. At the moment of secretion, pro-insulin is cleaved, releasing C-peptide and functional insulin in to the blood circulation (Figure 4.22). 

The molecular mass has units termed daltons (Da), after the famous English chemist John Dalton (1766-1848). Eor example, the molecular mass of insulin might be said to be about 6000 daltons. However, this designation does not add much to understanding the concept of molecular size and we shall delete the dalton unit in all that follows. 

The opposite page presents models of insulin, a small protein. The biosynthesis and function of this important hormone are discussed elsewhere in this book (pp.l60,388). Monomeric insulin consists of 51 amino acids, and with a molecular mass of 5.5 kDa it is only half the size of the smallest enzymes. Nevertheless, it has the typical properties of a globular protein. 

Insulin, a pancreatic hormone, is a specific antidiabetic agent, especially for type I diabetes. Human insulin is a double-chain protein with molecular mass around 6000 that contains 51 amino acids (chain A—21 amino acids, chain B—30 amino acids), which are bound together by disulfide bridges. 

Chromatographic or electrophoretic analysis of conventional insulins generally yields three major fractions or bands a, b and c). Fraction a contains high molecular mass material which can be removed from the product by additional recrystallization steps. The major components of fraction b are proinsulin and insulin dimers, while insulin, as well as slightly modified forms of insulin (e.g. arginine-insulin and desamido-insulin), are found in fraction c. 

The higher molecular mass contaminants in conventional insulin preparations include various proteases. Such preparations are generally maintained in solution at acidic pH values (often as low as pH 2.5-3.5). This minimizes the risk of proteolytic degradation of the insulin molecules, as contaminant proteases are inactive at such pH values. 

Figure 8.3. Chromatographic purification of recrystallized insulin on a Sephadex G-50 gel filtration column. Separation of high molecular mass proteolytic enzymes, as well as proinsulin and some very low molecular mass material, is obvious. Insulin elutes from the column as a single peak, hence the term single peak insulin ...

A solution prepared by dissolving 20.0 mg of insulin in water and diluting to a volume of 5.00 mL gives an osmotic pressure of 12.5 mm Hg at 300 K. What is the molecular mass of insulin ... 

To determine molecular mass, we need to know the number of moles of insulin represented by the 20.0 mg sample. We can do this by first rearranging the equation for osmotic pressure to find the molar concentration of the insulin solution and then multiplying by the volume of the solution to obtain the number of moles of insulin. 

Knowing both the mass and the number of moles of insulin, we can calculate the molar mass and hence the molecular mass ... 

Since its discovery, isolation, and purification in the early twentieth century, insulin has been administered to diabetic patients exclusively by injection until the recent introduction of inhaled insulin. Insulin possesses certain physiochemical properties that contribute to its limited absorption from the gastrointestinal tract, and requires subcutaneous injection to achieve clinically relevant bioavailability. With a molecular size of 5.7 kDa, insulin is a moderately sized polypeptide composed of two distinct peptide chains designated the A chain (21 amino acid residues) and the B chain (30 amino acid residues) and joined by two disulfide bonds. Like all polypeptides, insulin is a charged molecule that cannot easily penetrate the phospholipid membrane of the epithelial cells that line the nasal cavity. Furthermore, insulin monomers self-associate into hexameric units with a molecular mass greater than 30 kDa, which can further limit its passive absorption. Despite these constraints, successful delivery of insulin via the nasal route has been reported in humans and animals when an absorption enhancer was added to the formulation. 

Several peptide products used in the treatment of diabetes mellitus, in addition to insulin, are currently administered by subcutaneous injection and these drugs are candidates for development of nasal formulations. Glucagon-like peptide-1 (GLP-l)-related peptides stimulate the insulin response to glucose and diminish the release of glucagon after a meal. These effects diminish the excessive postprandial increase in glucose observed after a meal in persons with type 2 diabetes mellitus. GLP-1-related peptides must be administered by subcutaneous injection before meals in order to be effective. This requirement for injection before each meal is likely to impact the utilization of these products by persons with type 2 diabetes. Exendin-4 is a GLP-1-related peptide with a molecular mass of 4.2 kDa. The development of a GLP-1-related peptide nasal formulation containing an absorption enhancer would allow patients to scll-administer one of these drugs just before a meal without the need for a subcutaneous injection. 

Human Insulin contains 54 amino acids and has a relative molecular mass of 5808. This small globular protein will purify quite efficiently on stationary phases with 100 A mean pore size. 

Salmon Calcitonin has 32 amino acids and a relative molecular mass of 3432. This comparatively large peptide is in fact bigger than Insulin Try >200 A pore size - you might be surprised. 

The receptor, particularly the /3 subunit, is extremely sensitive to proteolysis [14] and can give rise to the apparent association of peptides of lower molecular mass being associated with receptor preparations. Nevertheless, there are indications, from both immunoprecipitation [15,16] and cross-linking [17] studies, that other distinct protein subunits may be associated with the insulin receptor. It is possible that these proteins represent species that are functionally or structurally capable of interacting with the insulin receptor, yet are not covalently attached to the receptor itself. Hence their association with the receptor would be expected to be easily disrupted by the manipulative processes used in purifying the solubilized receptor. The... 

There are distinct receptors for both IGF-I and IGF-II. The IGF-I receptor is similar in structure to that for the insulin receptor, having a disulphide bridge-linked subunit (a-j8)2 structure [49-52]. The a subunit has a molecular mass of 130 kDa which is capable of binding IGF-I. The 95 kDa j8 subunit of the IGF-I receptor, like that for the insulin receptor, exhibits a tyrosyl kinase activity. In marked contrast, however, the IGF-II receptor is a monomeric protein of molecular mass 220 kDa [53,54] with no known intrinsic activity. 

The search for physiological targets of action of tyrosyl kinases has been very unrewarding. No clear target for the action of insulin, other than the receptor itself, has been identified [61]. However, evidence has been presented which suggests that a membrane glycoprotein of molecular mass 110-120 kDa may provide a substrate for the receptor kinase. The identity and function of this species, however, remain to be elucidated [61,74],... 

A. Lane 1, Molecular weight markers lane 2, Purified final product. Molecular mass markers are Novex SeeBlue Pre-Stained Standards and range as follows (from top to bottom) Myosin, 250-kDa BSA, 98-kDa Glutamic dehydrogenase, 64-kDa Alcohol dehydrogenase, 50-kDa Carbonic anhydrase, 36-kDa Myoglobin, 30-kDa Lysozyme, 16-kDa Aprotinin, 6-kDa Insulin B chain, 4 kDa. 

The insulin receptor is synthesized as a preproreceptor of 1370 amino acids. The proreceptor (molecular mass 190 kDa on SDS-PAGE) is glycosylated (Kahn, 1985) in the Golgi region, proteolytically cleaved into mature a- and /3-subunits, and inserted in the plasma membrane (Fehlmann et al., 1982) or degraded. The half-life of the insulin receptor was determined to be 7-12 hours (Kahn, 1985). 

There may, however, be other related peptides which are as yet uncharacterized. For example, forms of rat IGF-II have been described with molecular masses of8700 and 16300 (M25) these are immunologically related to the characterized peptide of molecular mass 7500 (M5). A big IGF-II has also been reported in human spinal fluid and serum (H9). It is conceivable that these species are incompletely processed precursor firrms of IGF-II. In addition to such variants of known IGFs, variants of different charge have also been described. One, an acidic peptide (pi 4.8) first detected by its insulin-like activity, cross-reacts in two radioimmunoassays for SM-C/IGF-I, but not in an assay for IGF-II (H13). Finally, the existence of a fetal somatomedin has been postulated (H2), but whether this differs from IGF-II remains to be established. 

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