Eur. J. Biochem. 104, 469-478
`
`(1980)
`
`Temperature Behaviour of Human Serum Albumin
`
`Rolf WETZEL, Manfred BECKER, Joachim BEHLKE, Heidi BILLWITZ, Siegfried BoHM, Bernd EBERT, Harald HAMANN,
`Johannes KRUMBIEGEL, and Gunter LASSMANN
`Zentralinstitut fur Molekularbiologie der Akademie der Wissenschaften der DDR, Bereich Molekularbiophysik, Berlin-Buch
`
`(Received March 13, 1979)
`
`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 inter-
`molecular 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 tem-
`peratures 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 stabi-
`lizer. We distinguish in general two stages in the heat
`treatment of albumin. The first stage includes re-
`versible 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 revers-
`ibility, the influence of concentration and environ-
`mental conditions and the molecular mechanism of
`the action of the stabilizers and their relative effecti-
`veness under various conditions. Experiments per-
`
`Abbreviations. H + 'H exchange, hydrogen - deuterium ex-
`change; 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 ex-
`periments. The authors suggest that species de-
`specifications and desantigenization may only be
`obtained by a specific splitting of peptide bonds and
`not by changing the secondary structure. Aoki et al.
`[5] studied the termal denaturation of bovine serum
`albumin in the alkali pH range. The results obtained
`by gel electrophoresis show various components after
`the heat treatment at 65 "C. Brand and Anderson [6]
`described the influence of heating and fatty acids on the
`aggregate formation.
`The changes in the physicochemical properties of
`albumin, which have already been described in the
`literature, are partly contradictory. The reason is
`that on the one hand the results obtained imply
`method-specific information, for instance on the
`aggregational behaviour and the development of
`p structure [2,6 - lo], and on the other hand the con-
`
`

`

`470
`
`Temperature Behaviour of Albumin
`
`centration dependence of the effects have been largely
`neglected.
`We have, therefore, attempted to obtain more
`profound information on the changes in the physico-
`chemical properties of albumin during thermal treat-
`ment, in particular on the influence of the concen-
`tration and of the free - SH groups of albumin by the
`use of various physicochemical methods which also
`permit the carrying out of investigations in a larger
`concentration range. A later communication will
`report on possibilities of influencing structural alter-
`ations in the thermal treatment of albumin.
`
`MATERIALS AND METHODS
`Human serum albumin (commercial product, lyo-
`philized) from the Institut fur Impfstoffe (Dessau,
`G.D.R.) was used for the investigations. If not in-
`dicated otherwise, 0.1 M phosphate buffer, pH 6,
`containing 0.2 M NaC1, served as solvent. The deter-
`mination of concentration was performed by spectral
`photometry with II = 280 nm on the basis of an
`absorption coefficient of A t & = 5.8 [lo]. The samples
`used for electron spin resonance (ESR) measurements
`had been filtered through gel (Sephadex G-150).
`Mercaptalbumin was chromatographically prepared
`by the separation of albumin molecules by DEAE-
`Sephadex A-50 [l 11. The electrophoretic analysis
`showed, in agreement with sedimentation measure-
`ment, that the mercaptalbumin contained monomers
`and only a low content of dimers. The labels 2,2,4,4-
`tetramethyl - 1,2,3,4, - tetrahydro - 5,6 - benzo - y - car-
`boline-3-oxyl(I) and 3-maleimido-2,2,5,5-tetramethyl-
`pyrrolidine-1-oxyl(I1) have been used for the ESR
`investigations. Label I, being non-covalently bound to
`albumin, was added to the albumin solutions 10 min
`before the beginning of measurements. A 0.02 M
`ethanol solution of the label was prepared for that
`purpose in advance. The conditions of titration were
`chosen so that the ethanol concentration was 1 % or
`less.
`For the covalent labelling of Cys-34 with label I1
`the protein solution was incubated with a 50-times label
`excess at 15 'C for 30 min. Then the unbound label
`molecules were separated on Sephadex G-25. The
`number of - SH groups labelled could be determined
`by double integration of the ESR spectra obtained
`after labelling. Disc electrophoresis with polyacryl-
`amide gel was used for gel electrophoretic investi-
`gations.
`The circular dichroism (CD) measurements were
`performed on a dichrograph CD 185 (Roussel-Jouan).
`The measurement of temperature dependence was
`carried out in a hermetically sealed cell after achieving
`equilibrium, i.e. when a change in CD could no longer
`be found (this was the case after heating for 1 h).
`
`The accuracy of temperature measurements was
`f 0.2 "C. The structure contents (a-helices, p structures
`and residual structures Q) were calculated according
`to the curve-fitting method [12]. The CD basis spectra
`of Chen et al. [13] were used with the calculations. The
`-SH groups were determined by means of Ellman's
`reagent [14]. The ESR spectra were recorded with a
`Varian E3 spectrometer. An on-line computer (KRS
`4200) served for the spectral analysis [15]. The
`maximum sucrose concentration was 37.5 wjv in the
`determination of the rotational correlation time.
`The viscosity was measured by a rotating cartesian-
`diver viscometer and an Ubbelode viscometer.
`The infrared spectra of the solutions were measured
`by the Perkin-Elmer 180 infrared spectrometer 180.
`The albumin samples dissolved in 0.05 M 'H20 phos-
`phate buffer (pH 7.4) were heated in a thermostated cell
`and kept at the specified temperature until no further
`spectral change could be demonstrated in the range
`of amide I band (about 1 h). Infrared spectroscopic
`measurements on films were performed in connexion
`with the H -, 'H exchange measurements.
`H + 'H exchange kinetics were observed on thin
`films at a temperature of 25 "C and a relative humidity
`of 97 % from the decrease in intensity of the amide A
`band. The intensities of the amide A band were sub-
`sequently normalized to the amide I band. The in-
`strumentation used for the measurements has been
`described elsewhere [16]. Films of heated albumin
`solutions (0.5 - 1 h at 80 "C) and untreated albumin
`solutions were studied. For the film preparation salt-
`free solutions were used.
`The sedimentation behaviour of the albumin
`samples was studied in an analytical ultracentrifuge
`Spinco E with ultraviolet absorption optics, mono-
`chromator, and photo-electric scanner in the speed
`range of 17000 to 40000rev.jmin at 20°C. The
`albumin solutions of different concentrations were first
`heated to the specified temperature for 1 h and brought
`to a concentration of 1.4 mg/ml, if required, by
`dilution with phosphate buffer after cooling down.
`The concentration gradients were recorded using
`monochromatic light at /?. = 280nm. The sedimen-
`tation coefficients were calculated from the time
`shift of the gradients and corrected to standard con-
`ditions (water, 20 "C). The concentration portions of
`monomers and oligomers were calculated from the
`absorbance ratios of the gradients.
`
`RESULTS
`THERMAL DENATURATION
`CD Measurements
`There are four peaks presents in the CD spectra:
`at 1- = 262 nm and 268 nm (optically active transitions
`
`

`

`R. Wetzel, M. Becker, J. Behlke, H. Billwitz, S. Bohm, B. Ebert, H. Hamann, J. Krumabiegel, and G. Lassmann
`
`47 1
`
`4
`
`0
`
`-
`c - -50
`E
`N 5 -100
`- -
`
`-D
`
`m
`m V
`al
`
`20
`
`I
`40
`
`+
`
`I
`80
`
`I
`60
`t ("Ci
`Fig.1. Normalized CD melting curves of albumin using (0]220.
`(1) c = 0.5 mg/ml; (2) c = 0.05 mg/ml. The degree of renaturation
`after heating to t = 65 "C; t = 75 " C ; t = 90 "C and recooling to 20 "C is indicated by arrows. Insert (A): CD spectrum of albumin in the
`peptide chromophore region. (1) c = 0.5 mg/ml; (2) c = 0.05 mg/ml; path lengths = 1 mm and 10 mm respectively. Insert (B): CD
`spectrum of albumin in the region of aromatic chromophores. c = 2 mg/ml; path length = 10 mm
`
`-150 L
`4
`
`of the aromatic amino acids and disulphide bonds) and
`at 1 = 208 nm and 220 nm (peptide absorption range)
`(Fig. 1).
`The amplitude of these peaks do not show any
`major difference in their dependence on temperature.
`Below 60°C the melting curves measured at
`J. = 262 nm and 268 nm have a somewhat more
`pronounced decrease
`than
`those measured at
`J. = 208 nm and 220 nm, where the measurements
`in the temperature range are reflecting those changes
`on the albumin molecule which do not yet have any
`influence on the helix content and are reversible. The
`CD peak at 220 nm has been analyzed mainly for the
`present investigaton as the amplitude of this band is
`correlated with the helix content of the protein.
`We could find only a slight decrease of the CD
`amplitude up to 60°C; then a further more pro-
`nounced reduction follows between 65 "C and 80 "C.
`There is a degree of denaturation of about SO% at
`80 "C (Fig. 1). While the changes in the CD spectrum
`which may be obtained up to 65°C are completely
`reversible, the samples heated to 80 "C will be subject
`to an irreversible denaturation of about 40 % (Table 1).
`The structure contents of the renaturated samples
`heated to t D = 65 " c correspond approximately to
`those of the original values. The denaturation at 75 "C
`results in an increase in the residual structure contents
`at the cost of the a-helix content. The p structure
`contents will remain nearly constant, but increase,
`however, during cooling down at the cost of the
`a-helices (Table 1).
`
`ESR Measurements
`
`The spin labels I (non-covalently bound) and I1
`(covalently bound on the cysteine-34) were used to
`characterize the structural changes. The advantage
`of label I and label I1 as compared to other labels is
`that I [2] as well as I1 [17] has only one binding site
`on the albumin. When labelling with I1 one obtains
`an ESR spectrum consisting of two components
`(A and B in Fig. 2) (calbumin = 5 mgiml). There is 98 %
`of the bound spin labels in a structured environment
`(in Fig. 2 marked with A) at 20 "C. The remaining 2 %
`(B) correspond to an unfolded environment of the
`spin labels. From the temperature dependence of the
`ratio between the components A and B in the ESR
`spectrum the beginning of a more pronounced un-
`folding of the albumin molecule from 55°C can
`be seen (Fig. 3). This unfolding will be still reversible
`up to a temperature of 68 "C.
`It may be concluded from the expansion of the
`(+ 1) component of the spectral content A and the
`increasing distance of the components of A (2 A:, in
`Fig. 2) by 0.5 G as compared to the spectrum at 20 "C
`that particles of different molecular weight will be
`formed (increase of the rotational correlation time
`by the formation of larger particles). The t, value of
`73 "C (50 o/, content in an immobilized environment,
`Fig. 3) is in relatively good agreement with the values
`obtained from CD data. That means that the increase
`in label mobility by unfolding of the pocket containing
`the free - SH group of cysteine-34 [18] and the con-
`
`

`

`472
`
`Temperature Behaviour of Albumin
`
`Table 1. Degree of denaturation D, (at the temperature of denaturation tD), degree of irreversible denaturation I, (measured after heating to the
`temperature of denaturation tD and subsequent cooling down to 20 "C), and structure contents (a, B, e) at different temperatures i n with the mean
`the curve-fitting results
`standard deviations of
`D = denatured; R = renatured
`
`Albumin
`Albumin + MalNEt
`Albumin
`Albumin + MalNEt
`Albumin
`Albumin + MalNEt
`Mercaptalbumin
`Mercaptalbumin + MalNEt
`Mercaptalbumin
`Mercaptalbumin + MalNEt
`Mercaptalbumin
`Mercaptalbumin + MalNEt
`
`"C
`
`20
`20
`65
`65
`75
`75
`20
`20
`65
`65
`75
`75
`
`-
`-
`12
`10
`45
`30
`-
`-
`12
`10
`38
`20
`
`-
`-
`0
`0
`30
`15
`-
`-
`0
`0
`22
`0
`
`61.1 f 0.4
`69.9 & 0.5
`54.9 f 0.5
`60.0 f 0.6
`33.6 f 0.4
`50.8 f 0.5
`66.3 f 0.5
`68.3 f 0.6
`60.0 f 0.8
`62.8 f 0.8
`43.6 f 0.7
`57.0 f 0.8
`
`22.2 f 1.5
`24.9 f 1.9
`16.3 f 2.0
`18.0 f 2.2
`22.6 f 1.5
`21.8
`2.1
`11.0 f 2.4
`7.0 f 2.4
`7.2 f 2.9
`7.0 f 3.2
`10.4 f 2.9
`6.5 f 3.2
`
`16.7 f 1.4 -
`5.3 f 1.9 -
`28.8 f 1.9
`59.6 f 0.3
`22.5 f 2.1
`66.7 f 0.5
`43.8 f 1.4
`40.8 f 0.4
`27.4 f 2.0
`61.8
`0.5
`22.7 f 2.3 -
`24.7
`2.4 -
`68.5 k 0.9
`32.8 f 2.8
`68.1 f 0.6
`30.3 f 3.1
`53.4 f 0.6
`46.0 f 2.8
`36.5 f 3.1
`68.6 f 0.6
`
`-
`-
`24.7 f 1.2
`21.5 f 2.0
`28.1 f 1.7
`29.4 f 2.0
`-
`-
`3.3 f 3.6
`11.9 f 2.2
`18.8 f 2.2
`10.9 + 2.4
`
`-
`-
`15.8 & 1.2
`11.8 k 2.0
`31.0 f 1.6
`8.7 f 2.0
`-
`-
`28.1 f 3.5
`20.2 f 2.1
`27.8 f 2.2
`20.5 f 2.3
`
`(A)
`
`Fig. 2. ESR spectrum of albumin lubelled with label II. A, strongly immobilized spectral components; B, weakly immobilized spectral com-
`ponents. Insert: schematic drawing of the different environments of label I1 bound to albumin according to A and B
`
`t
`
`5 -- I
`
`-.
`
`\
`
`"'$
`
`formation contents measured by CD will take place
`in parallel, the relative final values obtained, however,
`being different. A degree of denaturation of the pro-
`tein of about 45",, taken from CD measurements
`(Table 1) corresponds to an unfolding rate of about
`90% near the label. The cooling down of samples to
`20 "C after heating to temperatures of 68 "C or more
`with incubation times of 5 and 20min will result
`in different degrees of irreversible structural changes
`(Fig.3). In addition the temperature effect was also
`observed by means of the probe I (Fig. 3). The mobile
`spectral content of 2 % at 20 "C is due in this case to
`the spin label molecules being free in solution.
`The increase in the content of free spin label
`molecules, which may be found with a temperature
`rise, can be explained by a decreasing structurization
`of the binding area of the label on the protein. The
`same t, value of 73°C may be obtained when using
`the probe I and the label I1 as well (Fig.3).
`
`30
`
`40
`
`60
`
`70
`
`80
`
`0
`0
`
`10
`
`20
`
`50
`f ("CI
`Fig. 3. Temperature dependence of the ESR spectral component A ( %)
`of label II covalently bound to albumin ( x ). The arrows indicate the
`reversibility after different heating times. Temperature dependence
`of the ESR spectral component A of label I (%) noncovalently
`bound to albumin (0)
`
`

`

`R. Wetzel, M. Becker, J. Behlke, H. Billwitz, S. Bohm, B. Ebert, H. Hamann, J. Krumbiegel, and G. Lassmann
`
`413
`
`I
`i
`i
`
`a
`
`I
`
`X'
`
`68 69
`
`1 0 2 0 3 0 4 0 5 0 6 0 7 0
`t (OC)
`Fig.4. Determination of the A,, values (2 AJ
`peratures, using McConnell's method [lY]
`
`0
`
`at d ~ e r e n t tem-
`
`The change in polarity around the NO was ob-
`served by means of label I (Fig.4). With rising tem-
`perature the interaction between the spin label and
`the albumin molecule (according to the van der
`Waal's interaction) becomes more intensive and the
`NO group comes into stronger interaction with apolar
`areas. At 60 "C the line width changes as the rotational
`movement grows faster so that a further determination
`of this parameter is no longer possible.
`
`INFLUENCE OF ALBUMIN CONCENTRATION
`ON THE TEMPERATURE BEHAVIOUR
`CD Measurements
`The t, value for the lower concentration (0.05 mg/
`ml) is higher than that for the higher concentration
`(0.5 mg/ml) (Fig.1). The difference (At,)
`is 5°C.
`Albumin concentrations of 0.05 mg/ml and 0.5 mg/ml
`yield different CD curves (Fig. 1). The structure
`contents are also different for both the concen-
`trations. We determined the following structure con-
`tents at 20 "C and an albumin concentration of 0.05 mg/
`ml: a-helices = 52%; p structures = 6%; residual
`structures @ = 41 %. The a-helix content with Calbumin
`= 0.05 mg/ml is about 15 % lower than with Calbumin
`= 0.5 mg/ml.
`After heating to 90 "C (we may heat up to 90 "C at
`Calbumin = 0.05 mg/ml without any visible turbidity)
`and cooling down to 20 "C we have: a = 41 %;
`/3 = 12Xandg = 48%.
`It appears to be essential that in addition to the
`helix contents the p structure contents are also lower
`than with Calbumin = 0.5 mg/ml. This is certainly due
`to a lower degree of aggregation with less inter-
`molecular p structure contents (see Table 1). While
`
`c (mM)
`of albumin as u function of protein con-
`Fig. 5. Dynamic viscosity
`centration. Insert : linear region for low protein concentrations
`
`no turbidity of the solution could be found with
`albumin concentrations I 0.5 mg/ml at a temperature
`of 75 "C, a turbidity and precipitation were observed
`at the same temperature with Calbumin = 5 mg/ml, so
`that ultraviolet optical investigations in this range of
`concentrations at such high temperatures are no longer
`possible. This can be demonstrated by CD mea-
`surements at l. = 268 nm when a higher concen-
`tration can be used because of the relatively low
`absorption. With Calbumin = 2 mg/ml, after decreasing,
`the CD values begin to increase at 78"C, indicating
`increasing turbidity. At 82 "C an apparent CD value
`will be a attained which corresponds to the original
`value at 20 "C.
`The degree of irreversible denaturation does not
`exceed 40 % in any case investigated. When stabilizers
`are used (M. Zinke, M. Becker, R. Wetzel, unpub-
`lished results) the same final result will also be obtained.
`Gel electrophoretic investigation then shows only
`monomers and higher aggregates.
`Oligomers of different sizes could be found at
`temperatures of 60 "C or more (but less than 80 "C)
`and c = 0.5 mg/ml.
`Mercaptalbumin, however, will precipitate by
`85 "C even when 2 mM sodium octanoate is used as
`stabilizer (M. Zinke, M. Becker, R. Wetzel, un-
`published results), as measured by gel electrophoresis.
`Without addition of sodium octanoate the samples
`with Calbumin = 5.0 mg/ml and Calbumin = 50 mg/ml
`will precipitate at a temperature of 75 "C.
`
`Viscometric Investigations
`We have to distinguish between a linear range of
`the concentration dependence in the viscometric
`behaviour up to 65 mg/ml (approximately 1 mM)
`
`

`

`474
`
`Temperature Behaviour of Albumin
`
`0 '
`0
`
`.
`
`"
`
`
`I
`0.5
`
`1
`1.0
`Calburnin (g/dl)
`Fig. 6. In qr,~/c.lburnin versus Colbuman after treatment at several tem-
`perafures. The extrapolation to calburnin = 0 gives the intrinsic
`viscosity [q]. As commonly used [q] is given in dl/g. The con-
`centrations of albumin used in the solutions subjected to heat
`treatment before dilution were: (a) 14.8 mg/ml; (b) 9.6 mg/ml;
`(c) 5.8 mg/ml. Temperature of the treatment: (+) 20°C; (0, A, 0)
`60 "C; (0, A, W) 70 "C and (a) 80 "C. Albumin in the concentrations
`9.6 mg/ml and 14.8 mg/ml changes after treatment with 80°C to
`a non-measurable gel-like state
`
`
`
`I
`,
`1.5
`
`1700
`
`1600
`1650
`Wavenumber (cm-')
`Fig. 7 . Infrared spectra at dijJerent temperatures of albumin. The
`development of the shoulder in the amide I absorption band
`indicates the development of
`structures. After recooling the effect
`increases. tD = 75°C; c = 10 mg/ml; d = 0.05 mm. (-)
`20°C;
`.-) 75 T ; (----) 20 "C (after recooling)
`
`1550
`
`and an exponential one at higher concentrations
`(Fig. 5). The linear range was used to study the tem-
`perature influence. The instrinsic viscosity [v] of
`heat-treated albumin was determined with concen-
`trations of 5.8,9.6 and 14.8 mg/ml. The heat treatment
`was performed by an incubation of 60min at the
`respective temperature. The measurements were per-
`formed on the renatured albumin at 20 "C k 0.1 "C
`(Fig.6). For the determination of the intrinsic vis-
`cosity the quotient of the natural logarithm of the
`relative viscosity vrel and the albumin concentration
`c (In qrel/c) was plotted as function of the albumin
`concentration c (Fig. 6). The intrinsic viscosity remains
`constant in the range of concentrations studied up to a
`temperature of 60 "C and is consistent with the value
`found in the pertinent literature, [q] = 0.037 dl/g [20],
`within the error range (k 15 x).
`
`Infrared Spectroscopic Investigations
`The results of the CD measurements (Table 1)
`show the increase in the b structure contents of the
`albumin after heating and subsequent cooling down.
`The development of those b structures may be observed
`also by means of the infrared spectroscopy with higher
`concentrations. The shoulder in the amide I absorp-
`tion band at 1615 cm-' (Fig.7) after heat treatment
`indicates these changes in the secondary structure of
`albumin.
`
`The development of b structures in the thermal
`denaturation of albumin is dependent on the concen-
`tration. The shoulder in the absorption band can be
`found with a concentration of 50 mg/ml at a tempera-
`ture of 70 "C, with Calbumin = 1.4 mg/ml at 80 "C. The
`CD measurements performed with calbumin = 0.5 mg/
`ml did not indicate any increase in the fl structure
`contents at t = 75°C. An increase in the p structure
`contents could be found only after cooling down to
`room temperature (Table 1). The result obtained by
`the CD measurements, i.e. the further increase in the
`/3 structure contents upon cooling down after heating,
`could be verified by the infrared measurements. With
`a concentration of 10 mg/ml, where the development
`of b structures would be expected at lower tempera-
`tures than with the concentrations of 0.5 mg/ml used
`for the CD measurements, no shoulder was visible in
`the infrared absorption band at 66°C except after
`cooling down the samples.
`Infrared spectroscopic investigations on albumin
`films produced from heated and untreated solutions
`also show a heat-induced fl structure development.
`The amide A band of films produced from heated
`albumin solutions (80 "C) has a peak at 3285 cm-'.
`It indicated a low-frequency shift of 15 cm-' relative
`to the peak at 3300 cm-' in the film of untreated sam-
`ples and thus the development of p structures. A
`heat-induced development of ,8 structures may also
`be derived from H+'H exchange measurements on
`
`

`

`R. Wetzel, M. Becker, J. Behlke, H. Billwitz, S. Bohm, B. Ebert, H. Hamann, J. Krumbiegel, and G. Lassmann
`
`415
`
`the original concentration co and temperature t
`Table 2. Influence of
`on the development of aggregates and p structures of albumin
`
`co
`
`t
`
`Monomers Oligomers
`( M , z lo6)
`4.0-4.5 S
`26 - 36 S
`
`form in the
`infrared spectrum
`(0 = not measured)
`(1615 cm-')
`
`t
`
`mg/ml
`
`"C
`
`1.4
`1.4
`7.0
`50.0
`10.0
`50.0
`1.4
`7.0
`10.1
`7.0
`10.0
`1.4
`1.4
`0.46
`1 .o
`1.3
`1.4
`7.0
`10.1
`
`20
`65
`65
`65
`68
`68
`70
`70
`70
`73
`73
`75
`78
`80
`80
`80
`80
`80
`80
`
`%
`
`100
`100
`100
`61
`52
`0
`100
`69
`39
`32
`19
`50
`44
`52
`40
`37
`36
`9
`0
`
`0
`0
`0
`39
`48
`100
`0
`31
`61
`68
`81
`50
`52
`48
`60
`63
`64
`91
`100
`
`-
`
`-
`0
`-
`0
`0
`-
`
`0 +
`0
`0
`-
`0
`- +
`0 +
`+
`
`I
`0
`
`I
`1
`log c,/rng
`rn-'
`Fig. 8. Influence of temperature and albumin concentration (CO)
`on the equivalent monomerloligomer ratio
`
`,
`
`
`
`I
`2
`
`-1
`
`0
`
`1
`log c,/mg rn-1
`Fig.9. Influence of albumin concentration (co) on the partial con-
`centrations ((,) of oligomers (0) and monomers (.).
`(----)
`calculated values. Sample pretreatment at 80 "C for 1 h
`
`2
`
`films. Films of the albumin samples heated to 80°C
`show an exchange rate markedly delayed as could be
`expected in the case of a development of p structures
`PI].
`
`Sedimentation Measurements
`Albumin will sediment in the neutral pH range
`with sedimentation coefficients of 4.0-4.5 S. If the
`samples are heated to more than 60°C and cooled
`down again there will be found in addition to the native
`protein a faster sedimenting but heterogenous frac-
`tion with sedimentation coefficients of 26 - 36 S. The
`content of these aggregates in the solution will depend
`not only on the temperature but also on the concentra-
`tion of the heated samples (Table 2).
`The tendency to develop these high molecular
`weight complexes or oligomers of the albumin in-
`creases obviously with rising temperature and concen-
`tration. Assuming that the heated solutions will only
`contain monomers and oligomers or aggregates, so the
`conditions (t, c) where both components are present
`in a portion of 50 % may be taken from Fig. 8.
`Plotting the partial concentration ci of monomers
`and oligomers of the samples heated to 80°C as a
`function of the total concentration co in a log-log
`system clearly reveals a linear increase in the oligomer
`
`content with the concentration. We can calculate from
`this relation, with the temperature and concentration
`given, not only the contents of oligomers formed but
`also those of the monomers as shown in Fig.9.
`The analytic results of the monomeric and oligo-
`meric components are without doubt still dependent
`on the concentration at which the sedimentation be-
`haviour was tested. As can be seen from Fig.10 the
`content of monomeric albumin in a solution with a
`concentration of 10.1 mg/ml, which has been heated
`to 70°C for 1 h and diluted after cooling down, will
`further increase with decreasing concentration. That
`possibly means that monomeric molecules are bound
`to the aggregates partly by adsorption, and this inter-
`action will be reduced by dilution. A complete disso-
`ciation of the oligomers with dilution has to be ex-
`cluded as the formation of aggregates will probably
`occur while forming covalent disulphide bridges but
`also hydrophobic interactions and p structures.
`
`INFLUENCE OF THE FREE - SH GROUPS
`ON THE TEMPERATURE BEHAVIOUR
`It has been found [22], by using a series of spin
`labels of attachement groups with different lengths,
`
`

`

`1
`I --.-
`
`476
`
`1.0
`
`A
`
`I
`
`0.5
`01
`0
`
`t
`
`I
`0.5
`
`I
`1.0
`
`I ,
`1.5
`
`oligorners
`
`Temperature Behaviour of Albumin
`
`denaturation process. It may be supposed that the
`formation of intermolecular disulphide bridges is
`an increasingly likely cause of the formation of dimers
`and a further aggregation by unfolding of the environ-
`ment of the -SH group.
`The solvents used for the isolation of albumin
`(methanol and ethanol) will cause a loosening of the
`environment surrounding the -SH group even with
`20% mixtures. This may be a starting point for the
`occurrence of dimers, which could not be found in
`blood plasma (except for bisalbumeria). The compari-
`son of albumin and especially prepared mercapt-
`albumin did not show any essential difference in the
`temperature behaviour as with the label 11, indeed
`only the albumins which have a -SH group which
`may be labelled could be covered. On the other hand
`a rotational correlation time of 32 ns for the albumin
`is obtained by the label I1 being strongly immobilized
`by the protein [24]. If the mercaptalbumin is labelled,
`however, we can find a value of 16 ns for the rotational
`correlation time. This value is not in the range of
`32 - 63 ns, the values possible for an elongated ellip-
`soid with an axial ratio of 3.5: 1 [25].
`A possible explanation for the observed decrease
`of rotational correlation time could be that fatty
`acids will be eliminated in the preparation of mercapt-
`albumin. This could result in a movement of the three
`globular domains of the albumin molecules relative
`to each other. To study the possibility that conforma-
`tional changes of the protein are connected with
`disulphide bridges, we performed comparative CD
`measurements with albumin (0.45 free -SH group/
`molecule) with mercaptalbumin (0.9 free - SH group/
`molecule) and with albumin and mercaptalbumin
`using - SH groups blocked by MalNEt (Table 1).
`The degree of denaturation at 75 "C as well as the
`degree of irreversible denaturation (after recooling
`to 20°C) will be reduced by the binding of MalNEt.
`With mercaptalbumin + MalNEt even a complete
`renaturation will be obtained. The degree of denatura-
`tion at tD = 75 "C is only 20%. If this value was not
`exceeded, a complete renaturation could be obtained
`with all the samples studied. Aggregates could not
`be demonstrated in this case by gel electrophoretic
`investigation either; without MalNEt aggregates could
`be found. If the aggregation through the formation
`of disulphide bridges is inhibited by the addition of
`MalNEt, the conformational properties will be re-
`tained, i.e. the structure contents do not change.
`A difference between mercaptalbumin and albumin
`is worth noting: it may be seen from gel electrophoretic
`measurements that the mercaptalbumin does not yet
`form aggregates at t = 65 "C and 70 "C, but the al-
`bumin does. That correlates with the somewhat lower
`content of p structures of the mercaptalbumin (Table 1)
`and could mean that the aggregation takes place in
`this temperature range mainly through the formation
`
`0
`
`0.5
`
`1.0
`
`1.5
`
`c0 (rng/mO
`Fig. 10. Influence of dilution on the monomerloligomer ratio of
`albumin. Dependence of partial concentration of the (A) mono-
`and (B) monomer (.)
`mers (.)
`and oligomer (0) levels on albumin
`concentration. Albumin (10.1 mg/ml) was previously heated at
`70 'C for 1 h
`
`1
`
`100
`80
`
`40
`
`20
`0
`0 10
`
`I
`
`I
`20
`
`,';.*-
`I
`I
`50 60 x) 80
`30
`40
`T (min)
`total spin concentration of spin label I in the
`Fig. 11. Decrease of
`presence of albumin at 76°C
`
`+
`
`I
`
`that the -SH group of cysteine-34 is located in a
`pocket having a depht of about 1 nm. The presence
`of a pocket will also be verified by fluorescence in-
`vestigations [23]. Considering the total spin label con-
`centration of label I one can see that there is a marked
`reduction of the NO group of the spin labels beginning
`at temperatures of more than 60°C. For that reason
`we did not perform any renaturations tests with the
`spin label I. Fig. 11 shows the course of the reduction
`of the NO group of label I at a temperature of 76 "C.
`The same reduction test (Fig. 11) was performed with
`the - SH group blocked by MalNEt to clarify whether
`the - SH group exposed to more than 60 "C reduced
`the NO group of spin label I. There was no reduction
`found. That is another finding that the -SH group
`is hidden in a pocket at temperatures of 60 "C or less.
`The width of that pocket may be estimated on the
`basis of the dimensions of I and I1 to 0.7 nm. There
`is an increasing unfolding of the pocket containing
`the free - SH group of cysteine-34 during the thermal
`
`

`

`R. Wetzel, M. Becker, J. Behlke, H. Billwitz, S. Bohm, B. Ebert, H. Hamann, J. Krumbiegel, and G. Lassmann
`
`477
`
`of intermolecular /3 structures. Then, after unfolding
`of the pocket (t 2 75 "C), the formation of aggregates
`through disulphide bridges will start to develop re-
`presenting irreversible change