Comparison of protein structure in the crystal and in solution.  II.  Tritium-hydrogen exchange of zinc-free and zinc insulin

Praissman, Melvin; Ja, Rupley

doi:10.1021/bi00846a052

Cited by 48 publications

(21 citation statements)

References 41 publications

(42 reference statements)

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“…Dilution results in dissociation of higher oligomers (e.g., hexamers and tetramers), which are first-order kinetic processes. Insulin follows an EX2 mechanism of exchange as determined in 1968 [29]. On this basis, we assume that various steps in the reaction diagram follow an EX2 mechanism of exchange; exchange in which the rate-limiting step is the exchange of the exposed site.…”

Section: Kinetics Of Amide Exchange In Solutionmentioning

confidence: 99%

“…On the basis of earlier work with insulin [29] and our own work with calmodulin [15], we assume that kinetics for exchange can be classified in three groups: fast, intermediate, and slow. Applying the kinetic fit described in the Methods section allows us to obtain the number of hydrogens in each of the three groups (Table 3).…”

Section: Kinetics Of Amide Exchange In Solutionmentioning

confidence: 99%

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Application of SIMSTEX to oligomerization of insulin analogs and mutants

Chitta

Rempel

Grayson

et al. 2006

J. Am. Soc. Mass Spectrom.

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The propensity of various insulins and their analogs to oligomerize was investigated by mass spectrometric methods including measurement of the relative abundances of oligomers in the gas phase and the kinetics of H/D amide exchange. The kinetics of deuterium uptake show a good fit when the exchanging amides are placed in three kinetic groups: fast, intermediate, and slow. r-Human insulin, of the insulins investigated, has fewer amides that exchange at intermediate rates and more that exchange at slow rates, in accord with its higher extent of association in solution. We adapted PLIMSTEX (protein ligand interactions by mass spectrometry, titration, and H/D exchange) to determine protein/ligand affinities in solution, to determine self-association equilibrium constants for proteins, and to apply them to various insulin analogs. We term this adaptation SIMSTEX (self-association interactions using mass spectrometry, self-titration and H/D exchange); it gives affinity constants that compare well with the literature results. The results from SIMSTEX show that some mutants (e.g., GlnB13) have an increased tendency to self-associate, possibly slowing down their action in vivo. Other mutants (e.g., lispro and AspB9) have lower propensities for self-association, thus providing potentially faster-acting analogs for use in controlling diabetes. (J Am Soc Mass Spectrom 2006, 17, 1526 -1534

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Section: Kinetics Of Amide Exchange In Solutionmentioning

confidence: 99%

Section: Kinetics Of Amide Exchange In Solutionmentioning

confidence: 99%

Application of SIMSTEX to oligomerization of insulin analogs and mutants

Chitta

Rempel

Grayson

et al. 2006

J. Am. Soc. Mass Spectrom.

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show abstract

“…The aggregational behaviour of pork and beef insulins in both acid and neutral formulations has been discussed [4,5,[8][9][10][11][12][13][14][15][16][17][18][19][20][21]. In this paper, unless otherwise stated, it will be assumed that 'insulin' refers to these four insulin formulations only.…”

Section: Relevant Literaturementioning

confidence: 99%

“…One notable study [36] has defined stability as P(t) = po e-kt where t = time, Po = biological activity at t = 0, and k = e BT, where T = absolute temperature and B is a proportionality constant. Instability of insulin solutions at 25 and 37 ~ has been noted [19,23]. Whether denaturation of the hormone at elevated temperatures caused aggregation of the hormone is not clear from the literature.…”

Section: Temperaturementioning

confidence: 99%

“…At the time the above authors reported that the chemical nature of these cross-linkages had not been elucidated but evidently these aggregates were formed during the extraction and isolation of insulin under acid conditions and were present at low levels in all solutions unless further methods for their removal were implemented [26,28]. With respect to salting out of the hormone during processing it has been postulated that hypertonic salt concentrations may alter the conformation of the crystalline protein [19].…”

Section: Processingmentioning

confidence: 99%

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