Structural alterations of albumin, their dependence on concentration and the role of free -SH groups at thermal denaturation, as well as the reversibility of thermally induced structural changes, were studied. Application of various physical methods provides information on a series of structural parameters in a major concentration range. Apart from changes of the helix content, heat treatment gives rise to p structures which are amplified on cooling and which are correlated with the aggregation of albumin. With rising temperature and concentration the proportion of b structures and aggregates increases.At degrees of denaturation of up to 20';;; complete renaturation is possible in every case. The structure content is concentration-dependent even at room temperature. It may be that intermolecular interactions induce additional a-helix structures which are less stable, however, than the ones stabilized by intramolecular interactions. Unfolding of the pocket containing the free -SH group of cysteine-34 enables disulphide bridges to be formed leading to stable aggregates and irreversible structural alterations. Through binding of N-ethylmaleimide to free -SH groups, which blocks the formation of disulphide bridges, it is possible to prevent aggregation and irreversible conformational changes. At temperatures below 65 -70 "C, oligomers are formed mainly via intermolecular p structures.In the preparation of human serum albumin for clinical purposes its behaviour at different temperatures is of importance. The albumin is treated at 60 "C for about 10 h to inactivate the hepatitis virus. The structure should be largely retained of course in this treatment. Sodium octanoate or sodium octanoate + acetyltryptophan may be used as stabilizer. We distinguish in general two stages in the heat treatment of albumin. The first stage includes reversible structural alterations, the second one includes irreversible structural alterations, which may not necessarily result in a complete destruction of the ordered structure [l--31. Although a number of investigations are available on the problem of the thermal exposure of albumin, there are still open questions to be answered concerning in particular the nature of structural alterations, the limits of reversibility, the influence of concentration and environmental conditions and the molecular mechanism of the action of the stabilizers and their relative effectiveness under various conditions. Experiments perAbbreviations. H + 'H exchange, hydrogen -deuterium exchange; ESR, electron spin resonance; CD, circular dichroism; MalNEt, N-ethylmaleimide.formed on horse serum albumin by Zimmermann and Dittmar [4] showed that higher molecular weight components will be increasingly formed after heat treatment of 15 min at 100 "C. If the time of exposure is 60min or more, the aggregation products will decompose into low-molecular-weight fragments which are serologically inactive. They may still cause, however, severe shock reactions in anaphylaxia experiments. The authors suggest that species des...
The modification of the triple helical structure in aqueous gelatin solutions by changing pH and adding alkyl sulphates at 298 K and after rechilling the solution to 283 K was investigated by CD-measurement. At 298 K the triple helical content at the IEP of the gelatin has its maximum value. It is only weakly affected by adding sodium dodecyl sulphate (SDDS) at concentrations < 10 -4 M/dm 3. The unfolding of the triple helix affected by pH and SDDS is reversible by rechilling the solution. The triple helical content of gelatin solutions decreases at SDDS concentrations higher than 10 -4 M/dm 3. In all cases the decrease of the amount of triple helical structure is connected with an increase of the cis-configuration in single chains and leads to chain reversals. At sufficiently high SDDS concentrations 3-sheets are formed. These changes are thermally irreversible. Sodium decyl sulphate (SDS) has a more minor influence than SDDS except in the range of the c.m.c, of SDS. At sufficiently high SDS concentrations, 3-turns appear.
The structural behaviour of gelatins from different raw materials and manufacturing processes at thermal denaturation and isothermal dehydration and rehydration is investigated by CD. At both thermal denaturation and isothermal dehydration with all gelatins examined, the triple helix content decreases. Simultaneously, the appearance of cis peptide bonds is observed. At rehydration, a structural hysteresis occurs, the reconstitution of the triple helix structure being correlated with a decrease in the content of cis peptide bonds. The possibility of the formation of chain reversals upon destruction of the triple helix is discussed.
An instrument is described for the simultaneous measurement of circular dichroism at all wavelengths in a limited spectral range. A polychromator and a charge-coupled device (CCD), serving as multichannel sensor, are arranged behind the sample cell, which is located close to the entrance slit, in contrast to the arrangement of a monochromator before the cell and using a photomultiplier as radiation detector, as usual until now. The CCD with low-noise electronics is driven by the system clock of a microprocessing unit controlled by a quartz oscillator and works fully synchronously with modulation and acquisition cycles. This leads to a high suppression of noise and systematic deviations. An electro-optic modulator with approximately rectangular excitation voltage is used. Partial CD spectra over the range of 80 nm each down to 200 nm have been recorded. The detection of a smaller amount of substance is possible than with other modern commercial instruments such as a JASCO J-600, with the same signal-to-noise ratio.
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