Chemistry and Biology of Proteins - F. Haurowitz 1953

Proteins with Hormonal Activity
Insulin

Insulin, a hormone produced by the Pancreas, is rapidly degraded by the gland's Proteolytic Enzymes if the pancreas itself or its extract is stored for any length of time. The destruction of insulin by pancreatic proteolytic enzymes was the very reason for the failure of earlier researchers who attempted to obtain an active extract of this gland.

Active extracts were successfully obtained by Banting and Best [21], who inhibited Trypsin activity by using acidified ethyl alcohol as a solvent. Trypsin activity can also be prevented by adding picric acid to pancreatic extracts [22]. Insulin is stable in the presence of dilute acids.

¹ There is ample reason to believe that The Essence of thyroglobulin and thyroxine action lies in the activation of enzyme Functions. A number of enzyme systems, including many oxidative ones, are already known whose activity increases significantly upon the administration of THYROID Hormones. According to the hypothesis put forward by B. I. Goldstein and his coworkers, thyroxine activates enzymes by reducing the disulfide groups in their molecules to sulfhydryl groups. An increase in the number of detectable sulfhydryl groups following the administration of thyroid hormones to animals was established for Liver and Muscle Proteins (B. I. Goldstein, M. B. Ginzburg, E. A. Kolli, E. Yu. Milgram, and O. S. Sklovskaya, Biokhimiya, 11, 447, 1946). An increase in the number of these groups in D-Amino Acid Oxidase preparations after the administration of thyroidin to rats was confirmed by A. V. Azyavchik, who also demonstrated a simultaneous increase in The activity of these preparations (A. V. Azyavchik, Dissertation, Moscow, 1952). The enhanced activity of proteolytic and oxidative enzymes—many of which are so-called sulfhydryl enzymes—can explain a number of the physiological properties of thyroid hormones. — Ed. note.

Insulin crystals are obtained by dissolving crude preparations in acetic acid, followed by the careful neutralization of the solution with ammonia and pyridine [23, 24]. Crystallization proceeds more efficiently upon The addition of saponin [25]. One milligram of pure crystalline preparation contains approximately 20 international units of insulin. In some fish species, the islets of Langerhans form a separate organ within the pancreas. In these fish, insulin is found exclusively in this organ and is absent from the rest of the glandular tissue [26].

The Molecular Weight of dissolved insulin is approximately 48,000 [27, 28], though it varies depending on solution concentration and pH. At pH values below 4 and above 7.5, insulin molecules dissociate into smaller units [27] with a molecular weight of approximately 12,000. X-Ray Structural Analysis has shown that insulin crystals consist of three similar subunits; consequently, the equivalent weight of crystalline insulin is 36,000. During Electrophoresis, insulin behaves as a homogeneous substance [29]. The isoelectric point of insulin lies near pH 5.3–5.8 [28].

Studies on the Amino Acid Composition of insulin revealed that it has a very high sulfur content. Detailed analysis demonstrated that the insulin molecule contains approximately 12% cystine and Cysteine, 10% leucine, and 12% Tyrosine [30] (see Table 1). Another characteristic feature of insulin's amino acid composition is the presence of A large number of free amino groups, significantly exceeding the number of Lysine residues contained in the molecule. Following complete Amino acid analysis, the following formula was established for insulin: arg₂, his₄, lys₂, glu₁₅, asp₆, gly₇, ala₇, val₈, leu₁₂, ile₃, pro₃, tyr₉, phe₆, ser₆, trp₂, cys (SS/2)₁₂, ammonia₁₂ [31]. The amino groups of insulin evidently determine its physiological activity to a certain degree, as this activity is lost upon Treatment with formaldehyde [32].

Inactivation of insulin is also caused by certain reducing agents, such as cysteine [34], thioglycolic acid [33], or leukomethylene blue [35]. The action of these compounds is most likely associated with the Cleavage of disulfide groups, each of which is converted into two sulfhydryl groups [33]. Periodic acid exerts a similar effect on disulfide groups, but in this case, sulfonic groups (—SO₃H) are formed instead of sulfhydryl groups [36]. Treatment of insulin with sulfuric acid yields insulin esters, which retain a large portion of hormonal activity [37]. If crystalline insulin is subjected to the action of crystalline trypsin, its activity decreases only slightly. This is because trypsin action hydrolyzes only one or two peptide bonds per molecule [38]. At the same time, Pepsin and Chymotrypsin readily cleave insulin [39].

If insulin is alkylated with dinitrofluorobenzene (see p. 125) and subsequently hydrolyzed, each 12,000-molecular-weight unit of insulin yields two molecules of dinitrophenylglycine and two molecules of dinitrophenylphenylalanine. This indicates that each of these units is built from four peptide chains terminating in Glycine and phenylalanine residues [40]. Sanger [41] successfully isolated from insulin two polypeptide chains A, containing glycine as terminal residues, and two polypeptide chains B, terminating in phenylalanine groups. Treatment of polypeptide A with dinitrofluorobenzene yields the following smaller Peptides: DNP-glycylisoleucine (DNP denoting dinitrophenyl), DNP-isoleucylvaline, DNP-glycylisoleucylvalylglycine, and DNP-tyrosine. Polypeptide A contains no Arginine, Histidine, phenylalanine, or Threonine. Similar treatment of polypeptide B yields DNP-phenylalanylvaline, DNP-phenylalanylvalylaspartic acid, DNP-phenylalanylvalylaspartylglutamic acid, DNP-lysylalanine, and threonyl-DNP-lysylalanine. These data convincingly demonstrate that the insulin molecule is constructed from various peptides linked together by cystine Disulfide Bonds. Arginine and histidine, which constitute the majority of the basic Amino Acids in insulin (see Table 1), are found only in some of these peptides [42].

Insulin readily combines with divalent metals, particularly zinc, cobalt, and cadmium; the insulin-zinc compound crystallizes more easily than pure insulin [43]. Of great clinical significance is the ability of insulin to form complexes with protamines. The hormonal activity of such a complex declines much more slowly than that of pure insulin [44].

Insulin is fairly resistant to Denaturing Agents and is not denatured by organic Solvents or dilute acids; Denaturation also does not occur upon The formation of monomolecular layers [45]. When insulin is heated in a mildly acidic solution, inactive fibrils are formed, which subsequently reaggregate into insoluble, spherical structures [46]. Interestingly, this process is reversible, and insulin can be reactivated by treatment with alkalis [46]. Insulin lacks antigenic properties and species Specificity [47].

Insulin injection lowers Blood sugar levels and prevents glucose loss and The Development of acidosis in diabetics. The addition of insulin to surviving rat Diaphragm tissue leads to an increased uptake of glucose and pyruvic acid by the diaphragm [48]. This effect, however, cannot be observed using diaphragm extracts [49]. According to some authors, this latter circumstance indicates that insulin alters Cell permeability to substances involved in Carbohydrate METABOLISM [49].

When working with commercial insulin preparations, one must bear in mind that they occasionally contain impurities that cause initial hyperglycemia, i.e., an elevation in blood sugar levels [50].