Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.

Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.     

Understanding the role of internal lysine residues of serum albumins in conformational stability and bilirubin binding

The role of internal lysine residues of different serum albumins, viz. from human, rabbit, goat, sheep and buffalo (HSA, RbSA, GSA, SSA and BuSA), in conformational stability and bilirubin binding was investigated after blocking them using acetylation, succinylation and guanidination reactions. No significant change in the secondary structure was noticed whereas the tertiary structure of these proteins was slightly altered upon acetylation or succinylation as revealed by circular dichroism (CD), fluorescence and gel filtration results. Guanidination did not affect the native protein conformation to a measurable extent. Scatchard analysis, CD and absorption spectroscopic results showed marked reductions (5-21-fold decrease in K(a) and approximately 50% decrease in the CD Cotton effect intensity) in the affinity of albumins for bilirubin upon acetylation or succinylation whereas guanidination produced a small change. Interestingly, monosignate CD spectra of bilirubin complexed with GSA, SSA and BuSA were transformed to bisignate CD spectra upon acetylation or succinylation of internal lysine residues whereas spectra remained bisignate in the case of bilirubin bound to acetylated or succinylated derivatives of HSA and RbSA. When probed by CD spectroscopy, bilirubin bound to acetylated or succinylated derivatives of GSA and SSA rapidly switched over to native albumins and not vice versa. These results suggested that salt linkage(s) contributed by internal lysine residue(s) play an important role in the high-affinity binding of bilirubin to albumin and provide stability to the native three-dimensional conformation of the bound pigment. Chloroform severely decreased the intensity of both positive and negative CD Cotton effects of bilirubin complexed with acetylated or succinylated derivatives of all albumins which otherwise increased significantly in the case of bilirubin complexed with native and guanidinated albumin derivatives, except the bilirubin-RbSA complex which showed a small decrease in intensity. These results suggest that the presence of salt linkage(s) in bilirubin-albumin complexation is(are) crucial to bring about effective and efficient stereochemical changes in the bound pigment by co-binding of chloroform which seems to have at least one conserved binding site on these albumins that is shared with bilirubin.     

Role of salt bridge(s) in the binding and photoconversion of bilirubin bound to high affinity site on human serum albumin

The role of salt bridge(s) (between epsilon-NH(2) groups of lysine residues of human serum albumin (HSA) and carboxyl groups of bilirubin) in the binding and photoconversion of bilirubin bound to high affinity site on HSA was investigated by covalent modification of approximately 20% internal (buried) lysine residues of HSA with acetic anhydride, succinic anhydride and O-methylisourea and white light irradiation of their complexes with bilirubin. The different HSA derivatives, namely, acetylated HSA (aHSA), succinylated HSA (sHSA) and guanidinated HSA (gHSA), thus obtained, were found to be homogeneous with respect to charge and size and characterized in detail in terms of mean residue ellipticity, Stokes radius, tryptophan fluorescence, bilirubin binding and the photochemistry of their complexes with bilirubin. All the three derivatives retained helical contents and molecular size (Stokes radius) similar to HSA except for sHSA which showed a slight increase in the Stokes radius from 3.56 to 3.64 nm. Further, fluorescence properties of aHSA and sHSA were also found to be different from HSA and gHSA. Based on difference spectral change, fluorescence quenching and fluorescence enhancement results of bilirubin bound to HSA and its derivatives, nearly 46 and 48% reduction in bilirubin binding was observed in the case of aHSA and sHSA, respectively. Both aHSA and sHSA showed a decrease of 8- and 10-fold, respectively, in association constant compared to native HSA. Although the bisignate circular dichroism (CD) spectra of an equimolar (1:1) bilirubin-HSA complex was retained by all three HSA derivatives, the intensity of both positive and negative CD Cotton effects decreased significantly in both aHSA and sHSA. gHSA which retained net charge identical to native HSA, showed little decrease in bilirubin binding and the intensity of bisignate CD Cotton effects. The photochemical reaction of bilirubin bound to aHSA and sHSA produced opposite results to those observed with HSA and gHSA. A brief (2 min) irradiation of an equimolar complex of bilirubin with both aHSA and sHSA accompanied a rapid shift (14-15 nm) in the absorption spectrum of the bound pigment towards the blue region and almost complete elimination of negative CD Cotton effects while only moderately affecting the magnitude of positive CD Cotton effects. On the other hand, similar treatment of the complexes of bilirubin with HSA and gHSA did not show any change in the absorption spectrum, only a slight decrease in the intensity of both positive and negative CD Cotton effects was observed. The fluorescence intensity of bilirubin bound to HSA and gHSA was increased upon irradiation with white light and after 30 min it was nearly twice the value observed at 0 min irradiation. Interestingly, no change in the fluorescence intensity of bilirubin bound either to aHSA or sHSA was observed upon irradiation, even on increasing the duration of irradiation to 1 h. Taken together, the results on fluorescence quenching, fluorescence enhancement, CD spectral changes and visible absorption spectroscopy suggest that salt bridge(s) of the type (-COO(-).(+)H(3)N-) in which the epsilon-NH(2) group(s) contributed by lysine residues, are not only involved in the enantioselective binding of bilirubin but also in the stereospecific photoisomerization of bilirubin bound to a high affinity site on HSA.     

Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding

In order to investigate the role of lysine residues in the interaction of bilirubin with bovine serum albumin, five succinylated preparations of albumin, namely: 23%, 39%, 49%, 55% and 87%, were prepared, and their conformational and bilirubin-binding properties were studied by the techniques of gel filtration, ultraviolet and visible spectroscopy, and fluorescence quenching. Gel filtration experiments performed at pH 7.0 and ionic strengths 0.15 and 1.0 suggested that the albumin molecule undergoes gradual disorganization with increase in succinylation. The Stokes radius and frictional ratio at ionic strength 0.15 increased from 3.7 nm and 1.36, respectively, for the native protein to 6.3 nm and 2.26 for maximally (87%) succinylated albumin. Interestingly, increase in ionic strength to 1.0 caused significant refolding in succinylated preparations as evidenced by a decrease in Stokes radius and frictional ratio (5.3 nm and 1.90 for 87% succinylated albumin). Progressive succinylation produced a steady decline in the intensity of bilirubin-induced fluorescence quenching, and in the visible spectral changes of the bilirubin-albumin complex at 480 nm. Both of these changes had a good correlation with increase in Stokes radius. Increase in ionic strength to 1.0 produced a significant reversal in these properties. From these results we conclude that probably none of the surface lysine residues is involved in bilirubin-albumin interaction, and that if lysine residues are involved in this interaction they must be buried in the protein interior.     

Changes in conformation and immunological activity of ovalbumin during its modification with different acid anhydrides.

In order to probe the cause and nature of conformational changes induced by the chemical modification of amino groups in proteins, five acylated derivatives of ovalbumin namely 21% acetylated, 32% succinylated, 90% butyrated 92% succinylated, and 95% acetylated ovalbumins were prepared and their molecular and immunological properties were systematically investigated. As evidenced by the ultraviolet difference spectral, solvent perturbation, gel filtration, and viscosity data, acylation of the amino groups produced a definite conformational change in native ovalbumin whose extent was higher for higher degrees of chemical modification. The solvent pertubation data showed an exposure of 0.5 tryptophan and 3 tyrosine residues in native ovalbumin; the exposure increased to 1 tryptophan and about 5 tyrosine residues in the maximally modified proteins (i.e. 90% butyrated, 92% succinylated, and 95% acetylated ovalbumins). The Stokes radius (2.7 nm) and intrinsic viscosity (3.9 ml/g) of ovalbumin increased, respectively, to about 3.4 nm and 7.7 ml/g upon acylation of its 18 lysine residues; the intrinsic viscosity of 95% acetylated ovalbumin was 7.2 ml/g. The reduced viscosity of ovalbumin (4.2 ml/g) which remained unaltered on raising the pH to pH 11.2, increased to 7.9 ml/g on succinylation of 18 lysine residues. On raising the ionic strength from 0.15 to 1.0, the value decreased from 7.9 to 6.2 ml/g. These observations taken together with the fact that the intrinsic viscosities of 92% succinylated and 90% butyrated ovalbumins are identical, argue against the presently prevalent proposal that electrostatic effects alone are responsible for the disruption of native protein conformation during chemical modification. The immunological activity of ovalbumin towards rabbit anti-ovalbumin expectedly decreased with acylation of its amino groups but the three maximally modified ovalbumins retained 40% immunological activity. This taken along with the spectral and viscosity data showed substantial native structure (format) in the three maximally acylated derivatives. The rabbit antiserum against 95% acetylated ovalbumin did not cross-react with acetylated lysozyme and reacted poorly with the native and 92% succinylated ovalbumins suggesting that the antigenic make-up of the three maximally modified ovalbumins is different.     

Interactions of native and modified cytochrome C with a negatively charged reverse micellar liquid interface

Native and chemically modified cytochrome C were dissolved in sodium bis(2-ethylhexyl) sulphosuccinate (AOT)-oil-buffer microemulsions. The native cytochrome C contains 19 lysine residues, these groups were modified by 1) acetic anhydride or 2) succinic anhydride. At pH 8.4 the native, acetylated and succinylated proteins carry +8, –3 and –12 elementary charges, respectively. The phase behaviour of the microemulsion systems was found to be highly dependent on the charge of the proteins. Compared to a protein free system the native protein induces a L-2 phase separation at lower temperatures. The acetylated protein has a small effect on the temperature for the phase transition, whereas in the case of succinylated cytochrome C the phase transition takes place at higher temperatures. When dissolved in AOT microemulsions, the native cytochrome C has a perturbed tertiary structure, as indicated by loss of the 695 nm absorption band, while both the modified proteins retain the same optical properties when dissolved in an AOT microemulsion as in a pure buffer solution. The pertubed structure of the native cytochrome C was further investigated by testing the stability of the reduced form of the protein dissolved in the microemulsion media. The native cytochrome is unstable at W > 10, whereas the two modified proteins were found to be stable at all W-values investigated. The average location of the three proteins was determined by pulse radiolysis. The quenching rate constant of the hydrated electron depends upon the location of the probe in the reverse micelle; the succinylated protein is localised in the aqueous core of the reverse micelles, but both the native and the acetylated forms were found to be localised close to or at the AOT interface.     

Conformational destabilization of Bacillus licheniformis α-amylase induced by lysine modification and calcium depletion

Bacillus licheniformis α-amylase (BLA) was chemically modified using 100-fold molar excess of succinic anhydride over protein or 0.66 M potassium cyanate to obtain 42 % succinylated and 81 % carbamylated BLAs. Size and charge homogeneity of modified preparations was established by Sephacryl S-200 HR gel chromatography and polyacrylamide gel electrophoresis. Conformational alteration in these preparations was evident by the larger Stokes radii (3.40 nm for carbamylated and 3.34 nm for succinylated BLAs) compared to 2.43 nm obtained for native BLA. Urea denaturation results using mean residue ellipticity (MRE) as a probe also showed conformational destabilization based on the early start of transition as well as ΔG(D)(H(2)O) values obtained for both modified derivatives and Ca-depleted BLA. Decrease in ΔG(D)(H(2)O) value from 5,930 cal/mol (for native BLA) to 3,957 cal/mol (for succinylated BLA), 3,336 cal/mol (for carbamylated BLA) and 3,430 cal/mol for Ca-depleted BLA suggested reduced conformational stability upon modification of amino groups of BLA or depletion of calcium. Since both succinylation and carbamylation reactions abolish the positive charge on amino groups (both α- and ε- amino), the decrease in conformational stability can be ascribed to the disruption of salt bridges present in the protein which might have released the intrinsic calcium from its binding site.     

On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin: effect of epsilon-NH(2) groups blocking and chloroform binding

Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when epsilon-NH(2) groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal epsilon-NH(2) groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between epsilon-NH(2) groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR-HSA complex.     

Streptavidin in antibody pretargeting. 3. Comparison of biotin binding and tissue localization of 1,2-cyclohexanedione and succinic anhydride modified recombinant streptavidin

Recombinant streptavidin (rSAv) is of interest as a carrier of alpha-emitting radionuclides in pretargeting protocols for cancer therapy. Due to the inherently high kidney localization of rSAv, modification of this protein is required before it can be useful in pretargeting. Previous studies (Wilbur, D. S., Hamlin, D. K. et al. (1998) Bioconjugate Chem. 9, 322-330) have shown that succinylation of rSAv using succinic anhydride decreases the kidney localization appreciably. In continuing studies, the biotin binding characteristics and biodistribution in mice of rSAv modified by reaction with succinic anhydride (amine modification) or 1,2-cyclohexanedione (arginine modification) have been compared. Modification of rSAv was conducted using 5-50 mol equiv of succinic anhydride and 60-200 mol equiv of 1,2-cyclohexanedione. Most studies were conducted using rSAv modified with the highest quantities of reagents. Succinylation of rSAv did not alter binding with biotin derivatives, but a small increase in the biotin derivative dissociation rate was noted for arginine-modified rSAv. Amino acid analysis of 1,2-cyclohexanedione-treated rSAv indicated about 40% of the arginine residues, or an average of 1.6 residues per subunit, were modified, whereas none of the lysine residues were modified. IEF analyses showed that the pI of the arginine-modified rSAv was 5.3-6, whereas the pI for the succinylated rSAv was approximately 4. Electrospray mass spectral analyses indicated that one to three conjugates of 1,2-cyclohexanedione, and two to three conjugates of succinic anhydride, were obtained per subunit. Both modification reactions resulted in greatly decreasing the kidney localization of rSAv (normally 20-25% ID/g at 4, 24, and 48 h pi). However, the kidney concentration for the succinylated rSAv continued to decrease (5% ID/g to 1.5% ID/g) from 4 to 48 h pi, whereas the concentration (5% ID/g) remained constant over that period of time for the arginine-modified rSAv. In contrast to this, the liver concentration appeared to be slightly higher (3% ID/g vs 2% ID/g) at the later time points for the succinylated rSAv. When less than 50 mol equiv of succinic anhydride were employed in the modification of rSAv, a correlation between increasing kidney localization with decreasing equivalents reacted was observed. Although the differences in the two modified rSAv are not substantial, succinylated rSAv appears to have more favorable properties for pretargeting studies.     

A correlation between changes in conformation and molecular properties of bovine serum albumin upon succinylation

Using succinic anhydride, six succinylated derivatives of bovine serum albumin having percent modification in the range of 23-87% were prepared and their physicochemical and immunological properties were studied. Measurements of Stokes radius, frictional ratio, UV spectra, solvent perturbation, solubility, and immunological cross-reactivity against anti-bovine serum albumin antiserum revealed that the protein undergoes gradual changes in its native conformation with increase in the degree of succinylation. These changes were less marked below 50% modification but became pronounced above 50% modification. However, even the maximally modified preparation (87%) contained a significant amount of folded structure. Interestingly, though the measurements of various molecular properties revealed significant changes in 23-49% modified preparations, the solubility parameters for these preparations which were obtained at high ionic strength were indistinguishable from those of the native protein. The various results taken together suggest that at lower degrees of chemical modification, the conformational changes were produced mainly because of an increase in electrostatic free energy, whereas at higher degrees of modification, steric hindrance in addition to the electrostatic factor seems to make a substantial contribution to the conformational changes in the modified proteins.     

Kinetics of fatty acid binding ability of glycated human serum albumin

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.     

Apolipophorin III lysine modification: Effect on structure and lipid binding

Apolipophorin III (apoLp-III) from Locusta migratoria was used as a model to investigate apolipoprotein lipid binding interactions. ApoLp-III contains eight lysine residues, of which seven are located on one side of the protein. To investigate the role of positive charges on lipid binding, lysine residues were acetylated by acetic anhydride. The degree of acetylation was analyzed by SDS-PAGE and MALDI-TOF, indicating a maximum of eight acetyl additions. Modified apoLp-III remained α-helical, but displayed a decreased α-helical content (from 78 to 54%). Acetylation resulted in a slight increase in protein stability, as indicated by a change in the midpoint of guanidine-HCl induced denaturation from 0.55 (unmodified) to 0.65 M (acetylated apoLp-III). Lipid bound apoLp-III, either acetylated or unmodified, displayed similar increases in helical content and midpoint of guanidine-HCl-induced denaturation of ∼ 4 M. The ability to solubilize vesicles of dimyristoylphosphatidylcholine remained unchanged. However, the rate to solubilize dimyristoylphosphatidylglycerol vesicles was reduced two-fold. In addition, a decreased ability to stabilize diacylglycerol-enriched low density lipoproteins was observed. This indicated that lysine residues are not critical for the protein's ability to bind to zwitterionic phospholipids. Since binding interactions with ionic phospholipids and lipoproteins were affected by acetylation, lysine side-chains may play a modulating role in the interaction with more complex lipid surfaces encountered in vivo.     

Effect of succinylation (3-carboxypropionylation) on the conformation and immunological activity of ovalbumin.

The epsilon-amino groups of ovalbumin were modified with succinic anhydride; as many as 16 lysine residues were succinylated (3-carboxypropionylated). The five succinylated derivatives thus prepared were homogeneous with respect to the extent of chemical modification as shown by electrophoretic and immunological data. Succinylation of the amino groups altered electrophoretic mobility and isoionic pH of ovalbumin in the expected direction. U.v.-absorption and fluorescence spectra suggested changes in the microenvironment of the chromophores in the modified proteins. The difference-spectral results showed greater exposure of tyrosine and tryptophan residues in the succinylated ovalbumin. Increase in susceptibility to tryptic digestion, Stokes radius and intrinsic viscosity of native ovalbumin, which was observed on successive increase in the chemical modification, demonstrated a conformational change that was proportional to the extent of modification. The loss of immunological reactivity caused by chemical modification also indicated a conformational change in succinylated ovalbumin. The fact that the intrinsic viscosity of maximally modified ovalbumin was less than one-third of that for the completely denatured protein in 6M-guanidinium chloride suggested that the modified protein contained significant residual native structure. The latter presumably accommodates some antigenic determinants accounting for 37% residual immunological activity observed with maximally succinylated ovalbumin.     

Streptavidin in antibody pretargeting. 5. chemical modification of recombinant streptavidin for labeling with the alpha-particle-emitting radionuclides 213Bi and 211At

We are investigating the use of recombinant streptavidin (rSAv) as a carrier molecule for the short-lived alpha-particle-emitting radionuclides 213Bi ( t 1/2 = 45.6 min) and 211At ( t 1/2 = 7.21 h) in cancer therapy. To utilize rSAv as a carrier, it must be modified in a manner that permits rapid chelation or bonding with these short-lived radionuclides and also modified in a manner that diminishes its natural propensity for localization in the kidney. Modification for labeling with (213)Bi was accomplished by conjugation of rSAv with the DTPA derivative p-isothiocyanato-benzyl-CHX-A' (CHX-A'), 3a. Modification for direct labeling with 211At was accomplished by conjugation of rSAv with an isothiocyanatophenyl derivative of a nido-carborane (nCB), 3b, or an isothiocyanatophenyl-dPEG/decaborate(2-) derivative, 3c. After conjugation of the chelating or bonding moiety, rSAv was further modified by reaction with an excess (50-100 equivalents) of succinic anhydride. Succinylation of the lysine amines has previously been shown to greatly diminish kidney localization. rSAv modified by conjugation with 3a and succinylated rapidly radiolabeled with 213Bi (<5 min), providing a 72% isolated yield. 211At labeling of modified rSAv was accomplished in aqueous solution using chloramine-T as the oxidant. Astatination of rSAv conjugated with 3b and succinylated occurred very rapidly (<1 min), providing a 50% isolated radiochemical yield. Astatination of rSAv conjugated with 3c and succinylated was also very rapid (<1 min) providing 66-71% isolated radiochemical yields. Astatination of succinylated rSAv, 2a, which did not have conjugated borane cage moieties, resulted in a much lower radiolabeling yield (18%). The 213Bi or 211At-labeled modified rSAv preparations were mixed with the corresponding 125 I-labeled rSAv, and dual-label in vivo distributions were obtained in athymic mice. The in vivo data show that 213Bi-labeled succinylated rSAv [ 213Bi] 6a has tissue concentrations similar to those of 125 I-labeled modified rSAv [ 125 I] 6b, suggesting that (213)Bi is quite stable toward release from the chelate in vivo. In vivo data also indicate that the (211)At-labeled rSAv conjugated with 3b or 3c and succinylated are stable to in vivo deastatination, whereas succinylated rSAv lacking a boron cage moiety is subject to some deastatination. The modified rSAv conjugated with nido-carborane derivative 3b has a higher retention in many tissues than rSAv without the carborane conjugated. Interestingly, the rSAv conjugated with 3c, which also contains an m-dPEG 12 moiety, has significantly decreased concentrations in blood and other tissues when compared with those of direct-labeled rSAv, suggesting that it may be a good candidate for further study. In conclusion, rSAv that has been modified with CHX-A' and succinylated (i.e., 5a) may be useful as a carrier of 213Bi. The encouraging results obtained with the PEGylated decaborate(2-) derivative 3c and succinylated (i.e., 5c) suggests that its further study as a carrier of 211At in pretargeting protocols is warranted.     

Helix stability in succinylated and acetylated ovalbumins: effect of high pH, urea and guanidine hydrochloride

Previous studies (Batra, P.P., Roebuck, M.A. and Uetrecht, D. (1990) J. Protein Chem. 9, 37-44) showed that succinylation or acetylation of 75% of the lysine residues has little effect on the secondary structure of ovalbumin. The acylation of the remaining 25% lysine residues, which apparently are partially buried, results in a substantial loss of the helical structure. These conformational changes may be due not only to electrostatic repulsions introduced by succinylation or acetylation of the positively charged epsilon-amino groups but also to steric hindrance, since an increase in the ionic strength failed to reverse the loss of the helical structure. An increase in pH to 12.2 results in a complete helix-to-coil transition in the maximally succinylated ovalbumin (but not in the partially succinylated or in any of the acetylated ovalbumins including the maximally acetylated derivative), perhaps because it is most expanded and its molecular interior most accessible to solvent as succinylation replaces +1 charge of epsilon-amino group with a -1 charge so that a net of -2 charge per succinyl group is placed on the protein molecule. This helix-to-coil transition in the maximally succinylated ovalbumin induced by high pH is fully reversed by increasing the ionic strength, indicating that only electrostatic effects are responsible for this disruption. Studies have also shown that although there is no loss of the helical structure until after the 75% surface lysine residues have been acylated, the helical structure does become progressively destabilized with increasing degree of modification, a conclusion drawn from urea unfolding curves. This destabilization of the helical structure is due primarily to electrostatic effects, as an increase in the ionic strength led to an increase in the urea transition mid-point. Unlike urea, the guanidine hydrochloride unfolding curves indicate that the transition mid-point for the native protein, as well as for the maximally succinylated and acetylated derivatives, is about the same, perhaps because the denaturant itself acts as an electrolyte.     

Fluorescein conjugates of 9- and 10-hydroxystearic acids: synthetic strategies, photophysical characterization, and confocal microscopy applications

Different strategies are presented to conjugate a fluorescein moiety to 9- and 10-hydroxystearic acids (HSAs). 5-Amino-fluorescein (5-AF) was used as a starting reagent. When reacted with acyl-chloride-modified HSAs, 5-AF gave rise to stable amide derivatives with a 75% reaction yield. These products exhibited the typical steady-state and time-resolved fluorescence properties of the fluorescein chromophore with absorption at 494 nm and emission at 519 nm. Flow cytometry studies confirmed the distinct proapoptotic effect of underivatized 9-HSA on Jurkat cells and revealed a comparable ability of its amide derivative. Confocal microscopy imaging studies showed that green fluorescence could stain intracellular membranous structures. Moreover, dual-dye labeling with Mito Tracker Red, followed by colocalization analysis, revealed that HSA can move to the mitochondria. Thus, fluorescent derivatives of HSA can be used to monitor the localization of these biologically active molecules in living cells and can provide a useful tool for linking biochemical investigation with optical visualization methods. In contrast, when unmodified HSAs were used, the reaction gave monoesterified and diesterified fluorescein derivatives. These products exhibited unusual steady-state and time-resolved fluorescence properties with the excitation wavelength at 342 nm and the emission wavelength at 432 nm. It is shown that the synthesized HSA amides of fluorescein provide all of the typical photophysical and instrumental advantages of this popular dye, whereas the unusual luminescence and excitation properties of the monoester and diester of the 5-aminofluorescein would make these dyes interesting to explore as potential candidates for two photon excitation applications.     

Noninvolvement of surface lysine residues of bovine serum albumin in bilirubin binding

Interaction of bilirubin with bovine serum albumin and its five succinylated forms was studied using fluorescence spectroscopy at two different ionic strengths i.e., 0.15 and 1.0 respectively. Affinity constant was found to be 1.8 x 10(7) litres/mole at 0.15 ionic strength which decreased to 4.4 x 10(6) litres/mole after 87% succinylation. On increasing ionic strength to 1.0, there was a slight decrease in affinity constant for native albumin. However, affinity constant remained same in 55 and 87% modified albumins at high ionic strength. These results suggest noninvolvement of surface lysine residues in bilirubin albumin complex.     

The role of the 6 lysines and the terminal amine of Escherichia coli single-strand binding protein in its binding of single-stranded DNA

Differential chemical modification of the lysines and amino-terminus of Escherichia coli single-strand binding (SSB) protein was used to determine their roles in the binding of SSB to single-stranded DNA (ssDNA). A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride. First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single-stranded ssDNA. Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride. Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry. The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA. Acetylation rate constants were determined from time course measurements. In the absence of ssDNA, the terminal alpha-amine of SSB was 10-fold more reactive than Lys residues at positions 43, 62, 73, and 87. The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition. In the presence of ssDNA, the reactivities of the amino-terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7-25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA. Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer-monomer interface.     

Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange (P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem.255, 10828–10833). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.