The accurate measurement of insulin molarity

Abstract

In order to study critically some physical characteristics and biological properties of insulin we needed an accurate measurement of the molarity of insulin solutions. Herskovits (1965) has measured the concentration ofinsulin by nitrogen analysis and published a value for the molar extinction coefficient of 5-96 x 103 at 277nm. This contrasts with the value of 5*22 x 103 obtained by Praissman & Rupley (1968) from dry-weight determinations corrected for counterion content. It occurred to us that this considerable difference could arise if the wrong value for the nitrogen content of insulin was assumed. The amide nitrogen atoms of insulin are known to be labile (Sundby, 1962) and commercial preparations ofinsulin have a degree ofheterogeneity that has been attributed to random loss of amide nitrogen (Carpenter & Chrambach, 1962; Mirsky & Kawamura, 1966). In an extreme case insulin could lose 6 amide nitrogen atoms/mol., thus decreasing its nitrogen content from 65 to 59 atoms/mol. (Sanger, 1960). Another potential source of error is the small but measurable amount of proinsulin that commercial preparations of insulin are known to contain (Schmidt & Arens, 1968). We have evidence that the most highly purified samples of insulin available from different commercial sources vary sufficiently in their amide nitrogen content to account for the high extinction coefficient obtained by Herskovits (1965), and we have established that the molar extinction coefficient for insulin is 5-53 x 103 at 277-5nm. We have also measured the molar extinction coefficient oftyrosine and find it to be 1-38 x 103 at 274-1nm. Ten-times-recrystallized bovine and porcine insulin from Novo Industri A.S (Copenhagen, Denmark), two different samples of six-timesrecrystallized bovine insulin from Boots Pure Drug Co. (Nottingham) and crystalline bovine insulin from Burroughs Wellcome Ltd. (Beckenham, Kent) were all generously supplied by the Companies concerned. L-Tyrosine was purchased from Sigma Chemical Co. (St Louis, Mo., U.S.A.) A.R. (NH4)2SO4 was dried and stored over P205 in an evacuated desiccator. Standard solutions of known nitrogen concentrations were made up by accurately weighing and diluting this dried salt.

Intrinsic Fibrillation of Fast-Acting Insulin Analogs

“…The pooled insulin concentration was determined using the extinction coefficient of 0.9521 ml/mg -1 /cm -1 at 277.5 nm absorbance. 40 The final concentration of each insulin analog was subsequently adjusted to 3.0 mg/ml with PBS buffer for most studies except as indicated. Buffer exchange on fresh analog samples was performed immediately prior to initiation of all fibrillation studies, and no bufferexchanged samples were stored for later use.…”

“…The absorbance of each supernatant solution at 277.5 nm was measured using a 1 cm path length quartz cuvette and a UV-Vis spectrophotometer, with soluble insulin concentrations determined by Beers Law using an extinction coefficient of 0.9521 ml/mg -1 /cm -1 . 40 Precipitate pellets were resuspended and washed with 1 ml water and recentrifuged, and the water wash was discarded. The washed pellets were frozen, dried under vacuum, and weighed to determine the mass of insoluble aggregates.…”

Identification of insulin variants using Raman spectroscopy

“…A UV wavelength of 254 nm was used for chromatographic detection (Milton Roy SpectraMonitor 3100). The concentrations of insulin in purified solutions were determined using a Cary 300 UV/Vis spectrophotometer with 1 cm quartz cell and a 280-nm detection wavelength [22]. For classification experiments, the insulin solutions were lyophilized after purification and redissolved in ultra pure water to concentrations of 100, 200, or 400 lM.…”

Generation of an acid-stable and protein-bound persulfide-like residue in alkali or sulfhydryl-treated insulin by a mechanism consonant with the .beta.-elimination hypothesis of disulfide bond lysis

“…Aliquots (0.5 mL) of the filtered solution were stored frozen at -20 °C. The precise concentration of freshly thawed solutions of insulin was determined from the absorbance at 277.5 nm by using the extinction coefficient of 5.53 X 103 M"1 cm"1 as determined by Harrison & Garratt (1969). Alkaline insulin solutions were prepared by the addition of KOH to a suitable amount of the insulin as prepared above.…”