Effects of Selective Biotinylation on the Thermodynamic Stability of Human Serum Albumin

Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA99) and 96% pure HSA (termed HSA96). According to the supplier, the 3% difference in purity between HSA96 and HSA99 is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations were modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA96, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA96-L and HSA96-H, with HSA96-L more stable than HSA99. The effect of multisite biotinylation is to stabilize Corresponding author.


Introduction
Human serum albumin (HSA) is the most prominent protein in plasma.Of the over 3000 different proteins that make up the plasma proteome, HSA has the highest relative concentration and comprises greater than 65% of the total protein by mass [2] [3].In vivo, HSA plays a major role as the principal cellular transporter in blood, and in doing so binds, both reversibly and covalently, to a broad spectrum of ligands of endogenous and exogenous origins.Binding and resulting modifications can be covalent, ionic or hydrophobic in nature [4] [5].Examinations of HSA samples derived from diseased blood have divulged a variety of chemical modifications, or adducts, of HSA that accompany human disease [6].Levels and types of covalent modifications of HSA can vary depending on the specific disease state(s) [7].
Because of the central role it serves in biochemistry, much is known about the structure, stability and function of HSA [2].HSA has served as a model for studies of protein folding and investigations of protein-ligand binding interactions [8].A number of studies have investigated the structure and thermal stability of HSA [1] [9]- [16].In these investigations a variety of spectroscopic techniques including Circular Dichroism, Infrared, and Fluorescence spectroscopy were employed.More recently (over the past 20 years) results of using Differential Scanning Calorimetry (DSC) to analyze the thermal stability of HSA have been reported [1] [9] [15] [17]- [20].
The primary structure of HSA is comprised of 585 amino acids residing on a single polypeptide chain.There are no tryptophan or methionine residues, but there is an abundance of charged residues such as lysine, arginine, glutamic acid and aspartic acid [2] [21] [22].HSA protein monomer has a molecular weight of ~66.5 kD and its 3-dimensional structure is a heart-shaped molecule composed of three domains, each with two sub-domains denoted A and B. The structure contains 17 disulfide bridges and one free sulfhydryl on cysteine residue 34 [2].The first 3-dimensional structure of HSA determined by X-ray crystallography was reported in 1992 [23] [24] followed by the higher resolution crystal structure [25].These and other studies have indicated that sites for ligand binding, and particularly binding sites of fatty acids, reside in the clefts between domains of the tertiary structure where there is an abundance of charged and polar residues such as lysine and arginine [26] [27].
A primary function of HSA is to transport fatty acid (FA).Consequently, a number of studies have investigated interactions and structural consequences of FA binding to HSA.These have identified the FA binding sites and, not surprisingly, some of these are associated with lysine residues [26] [27].Many of the same sites bound by FA are also likely targets for adduct formation or binding of other important ligands (i.e.therapeutic agents or diagnostic metabolites).It has been shown that FA binding can be allosteric and results in a conformational change of HSA.The FA bound conformation is apparently locked in by FA binding and conversion from the bound structure occurs with dissociation of FA [5].
Effects of FA on HSA thermodynamics were studied by one particular group [1] [15] who proposed, based on the observation of a bi-phasic thermal denaturation profile, the existence of two forms of HSA in the presence of long chain fatty acids.These were coined the defatted and un-defatted forms of HSA [1].Since our HSA 96 preparation also displayed a bi-phasic melting curve and contains 3% impurity specified by the supplier as "fatty acid and globulin", the possibility that our HSA 96 was analogous to the reported un-defatted form of HSA [1] is considered in our interpretation of the results.
In the current study our focus was on performing DSC analysis of HSA and quantitatively determining how the thermodynamic stability of HSA, an indirect measure of HSA structural integrity, is affected by covalent attachment of biotin to different sites on HSA 99 and HSA 96 .Through the choice of attachment chemistries, it was possible to selectively attach biotin to either a single site, or different multiple sites, and then make DSC mea-surements to assess the effects of specific site biotinylation on HSA thermodynamic stability.Biotinylation naturally occurs in several metabolic processes and it has been previously employed as a probe of conformational changes in proteins [28].Successful biotinylation of plasma has also been reported [29].For these reasons sitespecific biotinylation of HSA was conveniently chosen as the attachment moiety for investigating effects of covalent adduct formation on thermodynamic stability of HSA.

Protein Samples
Human serum albumin (HSA) was purchased from Sigma Aldrich (St. Louis, MO).Two different preparations of HSA with differing advertised purity were received as lyophilized powder.They were fatty acid and globulin free, ≥99% pure HSA (product number: A3872, lot number: SLBD740V) and ≥96% pure HSA (product number: A1653, lot number: SLBG2676V).The former was advertised as purified from the latter, but not necessarily the same batch.According to the supplier the 3% greater purity of the HSA 99 sample is due to the absence of slight amounts of FA and globulins purified away from HSA 96 .Hereafter, the 99%-pure and 96%-pure preparations of HSA are referred to as HSA 99 and HSA 96 , respectively.The latter is the well-known HSA fraction V isolated by the method of Cohn et al. [30].Although fraction V yields a large relative percentage of HSA, the preparation has been shown to be contaminated by ~2% globulins, primarily α 1 -globulins such as orosomucoid and long chain FA present at a Mole/Mole ratio of FA/HSA < 1.A typical value of this ratio = 0.51 was reported [2].Although an independent analysis of the specific contaminants in our HSA 96 preparation was not performed, the Certificate of Analysis of HSA 96 for our lot provided by the supplier reported not more than 2% of protein contaminant (as expected for fraction V).Thus, the majority of the remaining contaminant is nominally attributed to FA.Still the overall composition of the 3% contaminant of HSA 96 is not precisely known.To convey this fact, henceforth, the 3% impurity of HSA 96 is referred to as the "FA-and globulin-like contaminant" (FA/G-LC), with the belief that by mass the FA/G-LC is roughly comprised of 2% globulins and not more than 1% FA.In analogy several authors reported FA-free and FA-containing HSA species that have been referred to as defatted and un-defatted HSA [1] [15] [18].

Solvents and Reagents
Standard buffer solutions contained 10 mM potassium phosphate and 150 mM NaCl, pH = 7.4.Total ionic concentrations of buffers were verified by electrical conductivity measurements.After preparation and prior to use, buffer solutions were stored at 4˚C.All solutions and buffers were prepared with Nanopure deionized water.All chemicals and reagents were molecular biology grade or higher.

Solutions of HSA
Stock solutions of HSA were prepared at 1.0 mM by dissolving 0.067 g of HSA (67 kDa) in 1 mL buffer.Generally, reconstituted protein solutions were stored at 4˚C for at least 24 hrs before use.In the case of HSA 96 , samples that were somewhat unstable under storage were examined within 48 hrs after preparation.For DSC melting experiments, a portion of the HSA stock solution was diluted in buffer to a final concentration of 1.5 -2.0 mg/mL.

Biotin Attachment to Multiple Sites on HSA
Biotin was attached to multiple sites on HSA by targeting the primary amines of lysine residues.Of the 59 lysine residues in the primary structure it is believed that nearly 20 of these are likely to be sufficiently exposed, and therefore accessible for covalent attachment of Biotin in the 3-D structure of HSA [26].Biotin was attached to HSA using the EZ-Link Sulfo-NHS-Biotin kit (product number 21217 from Thermal Fisher Scientific) according to the supplier's instructions.For the attachment reactions, a 10 mM stock solution of Biotin was prepared by dissolving 3.2 mg Biotin in 500 µL of ddH 2 O.A solution containing a 1:1 molar ratio of HSA:Biotin was prepared by adding 3.02 µL Biotin stock solution per mL of a HSA solution at 2 mg/mL.In this manner HSA was incubated with various amounts of Biotin at a variety of molar ratios, HSA:Biotin, up to 1:50.For these reactions appropriate amounts of the Biotin stock solutions were added to HSA in the presence of the Bio-tin linking reagent and incubated for four hours at 20˚C.Generally, HSA samples prepared with different levels of attached Biotin were stored at 4˚C for at least 24 hours before removal of unattached free biotin.In this way Biotin was attached to both HSA 99 and HSA 96 preparations at a variety of HSA:Biotin ratios from 1:1 to 1:50.

Biotin Attachment to Single Sites on HSA
Biotin was covalently attached to HSA targeting the disulfides of cysteine residues.Presumably, under neutral conditions, only one reduced sulfhydryl is available for biotin attachment at cysteine-34 of the HSA structure [2], [31].In targeting the single reduced sulfhydryl at cysteine 34 of the HSA primary structure in these reactions, biotin was assumed to attach to only a single site on HSA.In attachment reactions HSA was incubated at 21˚C for four hours in the presence of increasing amounts of attachment reagent according to instructions provided by the supplier.For these reactions the EZ-Link Maleimide-PEG2-Biotin kit (product number 21902 from Thermal Fisher Scientific) was employed according to the supplier's instructions.Sample preparations were incubated at increasing molar ratios of HSA:Biotin, from 1:1 to 1:10.Attachment reactions were performed in 4 mL at a concentration of HSA = 1.6 mg/mL in the presence of increasing amounts of attachment reagent.In this way, Biotin was attached to both HSA 99 and HSA 96 preparations.

Removal of Unattached Biotin
When attachment reactions were complete, free (unattached) Biotin was removed using a Zebra spin column (product #A9892 from Thermal Fisher Scientific).In this procedure, 1.5 mL of each attachment reaction solution (total volume 3 mL) was added directly to a spin column, and the sample was washed three times with 2 mL buffer.The washed sample was then retrieved and pooled.

Determination of Protein Concentrations
Protein concentrations were determined at several steps.1) Prior to performing attachment reactions and; 2) After application of the Biotin removal kit, at each attachment ratio, prior to measuring DSC thermograms.Protein concentrations were determined using the BCA Protein Assay Kit (product #23225, Thermal Fisher Scientific).

Gel Electrophoresis
Samples were analyzed by electrophoresis on SDS polyacrylamide gels prior to, and after measurement of thermograms (not shown).Approximately 5 ug of protein sample was suspended in gel sample buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, 20 mM DTT pH = 8.3) and loaded onto a BioRad Any KD™ Mini-PROTEAN ® 15 well gradient polyacrylamide gel.Samples were heated at 95˚C for approximately 3 minutes before loading on the gel.Gels were run in gel sample buffer until the marker lane was well separated (35 minutes); then fixed in 50% methanol, 10% glacial acetic acid solution and stained with Coomassie Blue, then de-stained in 50% methanol, 10% glacial acetic acid; further soaked in 5% acetic acid solution, removed and photographed.The gel loading buffer for the samples contained 20 mM DTT to insure samples were in a reducing environment.

DSC Measurements
All DSC melting experiments were performed using a CSC Model 6100 Nano II-Differential Scanning Calorimeter (formerly Calorimetry Sciences Corporation, Provo UT, now TA Instruments).In a DSC melting experiment the excess heat capacity, ∆C p , is measured as a function of increasing temperature.Gasket tipped micopipettes provided an airtight means for protein/buffer injections and care was exercised in loading both buffer and sample solutions in the DSC to prevent introduction of air bubbles.The average of five buffer scans collected over the temperature range from 0 to 100˚C served as the buffer baseline for analyzing scans of protein samples.For all DSC experiments the temperature scan rate was 1˚C/min and the protein concentration was approximately 2 -2.5 mg/ml.To prevent degassing of solutions upon heating, all measurements were made at a pressure of 3.0 atm.Following each run, before a fresh buffer and sample were loaded in the DSC, sample and reference cells were carefully cleaned with a 95% ethanol solution.
Results of preliminary experiments (not shown) determined that the melting transition of HSA 99 spans the temperature region between 50 and 90˚C, and this is the temperature range for all displayed thermograms.To encompass this region, the temperature range used for measuring DSC thermograms of HSA 99 (and HSA 96 ) was from 45 and 95˚C.

DSC Data Analysis
DSC data was analyzed using the CpCalc 2.1 software package provided by the DSC manufacturer.The standard analysis procedure was comprised of six essential steps.1) Measured heat in µW was converted to raw molar heat capacity, ΔC p-raw (T) [cal/K•mol] versus temperature curves.2) These raw curves were then normalized for protein concentration (2.0 -2.5 mg/mL), molecular mass (66.5 kDa), partial specific volume of the protein molecules (0.733 cm 3 /g) [32] and sample cell volume producing normalized ΔC p (T) versus T curves (ΔC p-N (T) versus T). 3) The measured buffer baseline scan (ΔC p-buffer (T) versus T) curve was subtracted from the normalized (ΔC p-N (T) versus T) sample curve producing a buffer corrected, ΔC p-buffer corrected (T) versus T curve.4) Next, the baseline of the buffer corrected ΔC p-buffer corrected (T) versus T curve was determined using a polynomial function constructed by connecting the lowest and highest temperature points on the buffer corrected ΔC p-buffer corrected (T) versus T curve (generally the range from 45˚C -90˚C).5) This fitted baseline was then subtracted from the ΔC p-buffer corrected (T) versus T curve producing finally, a concentration normalized, buffer and baseline corrected ∆C p-N-buffer-baseline-corrected (T) versus T curve which is the DSC melting curve, or thermogram.
provided quantitative characterizations of the temperature-induced melting transitions of the HSA samples.Peak heights and peak widths at half height also served as an additional means of semi-quantitative comparison of thermogram shapes.
Analysis of DSC data in this fashion was performed under the simplifying assumptions that the melting transition of HSA occurs in a two-state manner, and that the overall change in the value of ΔC p at the initial and final temperatures is zero [33].Other than the choice of baseline and assumption that the ∆C p = 0, no other assumptions governed evaluation of the thermodynamic transition parameters.Results indicated the impact of these assumptions was not very severe, as evaluated parameters were in reasonable agreement with published results; and relative comparisons provided quantitative insight into effects of biotinylation on HSA structure and stability.Thus, we assumed the observed evaluated parameters corresponded to the denaturation transition of the HSA species in their standard state.The standard state thermodynamic parameters evaluated from thermograms of modified and unmodified HSA 99 and HSA 96 , i.e. generally denoted, ( ) , were employed to interpret their observed contrasting behaviors in response to covalent attachment of biotin.
More sophisticated models that include the possibility of multi-state transitions and a non-zero ΔC p have been applied to analyze DSC measured denaturation curves of HSA and provided evaluations of thermodynamic transition parameters [1] [13].Obviously depending on specific features of the particular analytical model employed to analyze DSC thermograms, actual values of the thermodynamic transition parameters obtained from the analysis could differ.However, relative values of the evaluated parameters should provide a more consistent basis for comparisons of results obtained using different models for thermogram analysis.

Melting Transitions of Unmodified HSA
The average of multiple measurements (>10) of the excess heat capacity, ΔC p , versus temperature (thermograms) for 99% pure HSA (HSA 99 ) and 96% pure HSA (HSA 96 ) samples, are shown in Figure 1.The distinctly different shapes of the thermograms for HSA 99 and HSA 96 in Figure 1 demonstrate the capability of DSC measure-Figure 1. Thermograms for HSA 99 and HSA 96 .The thermogram for HSA 99 (left curve, solid line) is single-peaked with t m = 63.3˚C.The curve for HSA 96 (right curve) is comprised of two overlapping major transitions with t m 's at 68.8 and 78.8˚C.Note, also a minor shoulder peak at 63.3˚C on the low temperature side of the HSA 96 main transition.Vertical error bars depict the variations in curve shape for multiple experiments on the same sample.The t m values vary on average 0.5˚C for multiple experiments.Thermograms of HSA 96 and HSA 99 (solid lines) were measured for samples prepared fresh and examined within a week of preparation.The dashed line is the thermogram for an HSA 96 preparation that was stored for three weeks at 4˚C. ments to discern effects on HSA of the presence of the FA-and globulin-like contaminant (FA/G-LC), advertised as comprising the 3% impurity in the case of HSA 96 .Error bars indicate the experimental variation from the average over at least three independent experiments.The curve for HSA 99 is the leftmost curve shown in Figure 1.As indicated by the error bars, in multiple DSC experiments the thermogram for HSA 99 was highly reproducible, with a nearly symmetric melting transition, a single major peak and slight high temperature asymmetry, and average t m = 63.3˚C± 0.2˚C.The peak width at half height of the HSA 99 thermogram is not more than 7˚C.The t m and curve shape of the HSA 99 thermogram are similar to those in published reports for defatted HSA also measured by DSC [1] [12] [17].These reports have onset of the HSA melting transition at around 50˚C with a span of 35˚C and completed by about 85˚C.Preparations of HSA 99 made from freshly diluted concentrated stock solutions (prepared from dry powder) and stored for over a year at 4˚C produced thermograms that were indistinguishable from those obtained from freshly prepared HSA 99 concentrated stock solutions.Accordingly, this stability of the HSA 99 thermogram confirms the structure of this preparation is quite stable during storage.
The average thermogram measured for HSA 96 is also displayed in Figure 1 (right-most curve).When the thermograms for HSA 96 , and HSA 99 are compared, the thermogram for HSA 96 is shifted to higher temperature by more than 5˚C and overall the transition seems to be comprised of at least two sub-transitions centered at 68.8 and 78.8˚C.There is also a very minor peak inflection on the low temperature side of the main transition at the same temperature as the t m of HSA 99 .Thermodynamic transition parameters evaluated from the thermograms of HSA 99 and HSA 96 are summarized in Table 1 and discussed later.
The bi-phasic melting transition observed for HSA 96 is remarkably similar to results of published DSC melting experiments on HSA in the presence of endogenous long chain fatty acid (LCFA) [1] [15].Measuring under nearly identical conditions (buffer and solvent, scan rate, protein concentration) these authors observed a biphasic melting transition attributed to coexistence of two different conformational states of HSA in the presence of LCFA.To indicate their relative affinity for binding LCFA these different forms were dubbed the "fatty acidpoor" or "defatted" and "fatty acid-rich" or "un-defatted" structures of HSA.Reportedly the bi-phasic melting transition resulted from redistribution of LCFA between the two forms, which differed in LCFA binding  affinity and temperature dependent stability, during the denaturation process.The observed similarity between our results and the published results [1] [15] leads to the supposition that the 3% impurity in the HSA 96 preparation (FA/G-LC) affects HSA structure and stability in a manner similar to that reported for LCFA [1].In support of this supposition, the reported relative amounts of LCFA present in the FA-poor (0.084 mole of LCFA per mole HSA) and FA-rich (1.43 mole LCFA per mole HSA) HSA preparations are comparable to ours and span the value of 0.51 mole of FA per mole HSA assumed for the composition of the 3% FA/G-LC in our HSA 96 preparation.
Because of the remarkable similarity between the thermogram for HSA 96 and published thermograms for defatted and un-defatted HSA measured under very similar solution and experimental conditions, the bi-phasic thermogram of HSA 96 in Figure 1 is also attributed to the presence of two protein populations.In analogy with previous reports [1] [15], and for reference in future discussions, the first peak on the HSA 96 thermogram at 68.8˚C is designated HSA 96-L ; while the second peak at 78.8˚C is referred to as HSA 96-H .The former is thought to more resemble the structure and stability of HSA 99 (but arguably not precisely the same).This distinction is evidenced by the small shoulder peak on the low temperature side of the HSA 96 thermogram attributed to the HSA 99 species.This small inflection occurs precisely at the t m of HSA 99 (63.3˚C), and likely indicates a slight amount of HSA 99 also present in the HSA 96 preparation.Presumably, both HSA 96-L and HSA 96-H bind FA to a lesser or greater extent, respectively [16].Similarity of the melting characteristics of the HSA 99 and HSA 96 preparations with published results also provided assurance of the fidelity and initial native state of these HSA preparations prior to covalent attachment of biotin.Before describing results of experiments on biotinylated HSA 99 and HSA 96 , several additional aspects of their behavior are described.

The Labile State of HSA96 and Conversion of HSA96 to HSA99
The HSA 96 preparation was not as stable in storage as HSA 99 .Examination of the HSA 96 thermograms at different times after preparation indicated the average HSA 96 thermogram displayed in Figure 1 was reproducible for at least one week (but not for much longer).On multiple occasions over the course of this study we observed the HSA 96 thermogram was not always reproducible for samples stored at 4˚C for longer than a week.
According to their thermograms, spontaneous conversion of HSA 96 to HSA 99 was observed.Accepting that HSA 96 contains a mixture of HSA 96-L and HSA 96-H the thermograms of HSA 96 (Figure 1) were found to differ for different preparations, and in particular to be quite sensitive to age of the sample (after those preparations) when the thermograms were measured.With this lability of the HSA 96 samples, reported thermograms for HSA 96 (displayed in Figure 1) were obtained for protein samples that were made from HSA 96 stock solutions prepared fresh from powder and examined within (at most) one week following preparation.Generally the time after preparation when thermograms were measured for HSA 96 and its biotinylated derivatives was 24 to 72 hours.
Although a systematic study of the state of the HSA 96 preparation at different times after preparation was not performed, typical results are shown in Figure 1.There, the thermogram of HSA 99 (freshly prepared and less than a week old, solid line) is compared with that obtained for a preparation of HSA 96 stored for three weeks prior to examination by DSC (dotted line).Clearly, the thermogram for the older HSA 96 is very similar to that of HSA 99 , evidencing conversion.Such conversion was observed in as little as one week for some HSA 96 samples stored at 4˚C.
Experiments investigating the reversibility of the HSA 99 melting transition were also performed (data not shown).For this investigation thermograms were measured for HSA 99 samples first heated in separate experiments to temperatures from 30 to 70˚C, cooled at the same rate, and then re-heated from 20 to 100˚C.Results showed when HSA 99 was first heated to temperatures up to 60˚C, just at the edge of the melting transition, cooled and re-melted, the thermogram was not different from the thermogram of unheated HSA 99 ; with a single peak, t m = 63.3˚C.This result was taken to indicate that up to about 60˚C, corresponding to early onset of the melting transition, the measured thermogram was unaffected by prior heating and cooling, and was reversible.In contrast, when the HSA 99 solution was pre-heated to temperatures above t m , i.e. up to 80˚C, cooled and re-melted, the observed thermograms for the pre-heated samples were very different from those observed for the unheated (or reversible) curves.These results were in concordance with those reported in published studies of HSA, that HSA melting is reversible up to ≈62˚C [12] [13].From experiments performed under conditions similar to ours these same authors found at temperatures above 75˚C, HSA resides in an irreversibly denatured, predominantly molten globule state.Reportedly, the rate of the irreversible reaction was considerably slower at lower temperatures (below 74˚C) [12].Under experimental conditions comparable to ours these authors asserted that even up to 74˚C the denaturation reaction is predominantly reversible.This is a major operating assumption.

Melting Transitions of HSA with Biotin Covalently Attached at a Single Site
Figure 2 shows the thermograms for HSA 99 and HSA 96 modified by biotinylation at a single site.Protein samples that produced the results shown in Figure 2 were prepared by using the Maleimide-activated reagent targeting the single reduced sufhydryl at cys-35 of the HSA primary structure.Products of the reactions were either HSA 99 or HSA 96 with biotin attached, denoted HSA 99-M(x:1) or HSA 96-M(x:1) , respectively (M = biotin attached with Maleimide).In Figure 2, thermograms are displayed for HSA 99-M(x:1) (a) or HSA 96-M(x:1) (b) incubated at molar ratios of biotin:HSA 99 = 1:1, 3:1 and 10:1 (x = 1, 3, 10).Error bars show experimental variation from the average curve (symbols) for HSA 99-M(x:1) at each incubation ratio.As seen in Figure 2(a), the effect of attachment on the HSA 99 thermogram is a peak height reduced by nearly 40% and shift of t m upward in temperature by over 5˚C.At x = 1 the peak width at half height increases by more than a factor of two.After these most dramatic changes initially at a 1:1 ratio, thermograms measured at biotinylation ratios of x = 3 and x = 10 are not greatly different.They have essentially the same t m with only slight increases in peak widths on the high temperature side; and peak heights are the same (within error).Results of these changes associated with biotin attachment also manifest in the measured thermodynamic parameters for HSA 99-M(x:1) summarized in show little variation between the different biotinylation ratios of x = 1, 3, 10.
In the same manner as for HSA 99 , biotin was covalently attached to HSA 96 (prepared fresh) and the thermograms were measured within 48 hours of sample preparation.Thermograms collected for the covalently modified HSA 96 are shown in Figure 2(b), and denoted HSA 96-M(x:1) .Effects of single site attachment of biotin to HSA 96 are dramatic, and there is a very clear difference between the HSA 96 thermogram and the HSA 96-M(x:1) Figure 2. Thermograms of HSA modified by biotinylation at a single site.Samples were prepared as described in the text using the Maleimide-activated reagent.(a) Thermograms for HSA 99 (solid line) and samples of HSA 99 with biotin attached denoted HSA 99-M(x:1) .Curves shown are for the HSA 99-M(x:1) samples prepared at attachment ratios of x = 1 (bold dash line), x = 3 (dotted line) and x = 10 (light dashed line).(b) Thermograms for HSA 96 (solid line) and HSA 96 with biotin attached, termed HSA 96-M(x:1) .Curves shown are for attachment ratios of x =1 (bold dash line), x = 3 (dotted line), and x = 10 (dash line).thermograms.Responses to biotin attachment at a single site on HSA 96 might be unexpected for several reasons.1) As indicated by the bi-phasic thermogram in Figure 1, the preparation of HSA 96 is comprised of two different forms that have been denoted HSA 96-H and HSA 96-L , present in their respective mole fractions.2) The effect of the biotinylation reaction on the relative population of these two states of HSA is unknown.3) The bi-phasic nature of the HSA 96 thermogram indicates HSA 96-H is more stable against denaturation than HSA 96-L .
Presumably, when the attachment reaction is performed using HSA 96 , both components, HSA 96-H and HSA 96-L are targeted for biotinylation.At incubation ratios of x ≥ 1, the two-peaked thermogram of HSA 96 in Figure 1 with t m 's at 68.8 and 78.8˚C is consolidated into a broad, single-peaked transition with an intermediate t m = 72.3˚C.The peak height is roughly the same as the major peak at 68.8˚C on the thermogram of HSA 96 alone (solid line).).The higher temperature peak corresponding to labeled HSA 96-H (HSA 96-H-M(1:1) ) seems to have shifted down a few degrees and become absorbed into the consolidated peak brought about by the up-shifted HSA 96-L-M(1:1) peak.The peak widths at half height of the thermograms for the HSA 96-M(x:1) samples in Figure 2(b) are reduced from that of HSA 96 (~20˚) to approximately 15˚C.Again as expected for a single biotinylation site, once saturation occurs (at x =1) incubation at increased ratios of biotin has little effect on the thermogram shape, or t m .In effect, there are no significant differences in the thermograms for HSA 96-M(x:1) at x ≥ 1.Just as observed for HSA 99-M(x:1) thermodynamic transition parameters for HSA 96-M(x:1) summarized in Table 1 are essentially the same at all attachment ratios examined.Although they show a similar response to biotinylation the actual thermograms of HSA 99-M(x:1) and HSA 96-M(x:1) are different.The peaks of the HSA 99-M thermograms are sharper with an average t m = 68.5˚Ccompared to the broader symmetric peaks of the HSA 96-M(x:1) thermograms with average t m = 73.8˚C.As shown in Table 1, and discussed in detail later, evaluated thermodynamic parameters for the thermograms of HSA 96-M(x:1) and HSA 99-M(x:1) also displayed different responses to biotin attachment.

Melting Transitions of HSA with Biotin Covalently Attached at Multiple Sites
The primary structure of HSA contains 59 lysine residues.Examination of the HSA crystal structure and results of cross linking studies suggested nearly half of the 59 lysine residues in HSA could be adequately exposed and therefore presumably accessible for biotinylation [26] [27].HSA samples that produced the thermograms shown in Figure 3 and Figure 4 were prepared by using the NHS-activated biotin reagent targeting the primary amines of lysine residues for attachment with increasing amounts of biotin.Thermograms of NHS biotinylated HSA 99 , denoted HSA 99-NS(x:1) , are shown in Figure 3. NS corresponds to biotin attached with NHS.Different thermograms shown in Figure 3 are for samples with increasing amounts of attached biotin.
Thermograms in Figure 3(a) are for HSA 99:NS(x:1) samples where the attachment ratios varied from x = 1 to 10. Figure 3(b) shows the thermograms obtained for HSA 99:NS(x:1) samples for which the incubation ratio varied from x = 10 to 50.Differences in the thermograms of HSA 99-NS(x:1) and HSA 99 seen in Figure 3 indicate a very clear effect of biotinylation.With increased attachment ratios from x = 1 to 20 there is a corresponding shift of the thermograms to higher temperature with incrementally increased t m .The peak heights of the modified thermograms are all about the same and reduced by about 15% from that of HSA 99 .Peak widths at half height of the modified thermograms increase by nearly 50% from the thermogram of HSA 99 alone.Figure 3(b) shows the thermograms obtained for HSA 99-NS(x:1) at incubation ratios from x = 10 to 50.Up to x = 20, the thermogram t m is shifted up in a titratable fashion by more than 9˚C.At higher incubation ratios examined (x = 40, 50) there are no further changes in t m .At incubation ratios greater than x = 10, the peak heights and widths at half height vary somewhat.Even with this variation, evaluated thermodynamic parameters in Table 1 are very consistent.
Thermograms of HSA 96 biotinylated at multiple sites are shown in Figure 4, and denoted HSA 96-NS(x:1) where again x is the incubation ratio corresponding to the relative amount of biotin attachment.Thermograms were measured for x = 1, 3, 5, 8, 10, 15, 20, 40, 50.A sampling of these is shown in Figure 4. Thermograms shown in Figure 4(a) were measured for HSA 96-NS(x:1) samples prepared at incubation ratios varying from x = 1 to 10. Thermograms in Figure 4(b) were obtained for HSA 96-NS(x:1) samples prepared at higher incubation ratios from x = 10 to 50.There is a stark difference between the HSA 96 thermogram (thin black line) and the HSA 96-NS(x:1) thermograms.Most notably, at the lowest attachment ratio (x = 1) the formerly two-peaked HSA 96 thermogram (thin black line in Figure 4(a)) becomes single peaked with t m = 66.2˚C (bold dash line Figure 4(a)).For x ≥ 1 the HSA 96-NS(x:1) thermograms have the same shape but are shifted up in temperature, with increased t m at higher attachment ratios up to about x = 40, with no changes thereafter (Figure 4(b)).
It is important to recall and consider that the initial preparation of HSA 96 from which the HSA 96-NS(x:1) samples were prepared was comprised of a mixture of two forms of HSA (referred to earlier as HSA 96-H and HSA 96-L ).Accordingly as seen in Figure 4 for HSA 96 the effect of biotinylation at lysine residues is different from that for single site biotinylation at cys-35 (Figure 2(b)).On the thermograms of HSA 96:NS(x:1) (Figure 4) there is no sign of the high temperature transition at 78.8˚C, denoted HSA 96-H , suggesting the attachment reaction itself must result in conversion of the originally present more stable structure, to a less stable one.The converted less stable form, HSA 96-L , when modified at a 1:1 ratio becomes HSA 96-NS(1:1) which is quite qualitatively similar to HSA 99-NS(1:1) , with an identical t m .Similar comparisons can also be made between HSA 96-NS(x:1) and HSA 99-NS(x:1) at increasing values of x (discussed later).Thermograms of HSA 96 samples incubated under the same conditions as the attachment reactions (four hours at 21˚C), without the attachment reagents, were indistinguishable from the thermogram of freshly prepared HSA 96 samples (not shown).
In summary, results in Figure 1 through Figure 4 clearly demonstrate the ability of DSC thermograms of HSA to detect the presence or absence of a slight amount of contaminant that contains globulins and FA, denoted earlier as FA/G-LC (Figure 1); detect changing states of HSA samples over time (Figure 1); differentiate covalent modification of HSA at a single site (Figure 2); or at multiple sites (Figure 3 and Figure 4).For both species of HSA, the effect of single site modification is a thermogram reduced in peak height with a small shift up in t m .For multiple site modification the thermogram displays a significant temperature shift up with increasing amounts of biotinylation.

Thermodynamic Transition Parameters
DSC measurements provided evaluations of the thermodynamic parameters of HSA melting transitions.  1 reveals differences between HSA 96-NS(x:1) and HSA 99-NS(x:1) .Differences between the two samples at lower attachment ratios up to x = 20 suggest the HSA 96-L-NS(x:1) samples at the lower attachment ratios reside in a different state than that of HSA 99-NS(x:1) .That is, HSA 96-L-NS(x:1) can also be bound by components of the FA/G-LC contaminant present in the HSA 96 preparation.The measured t m 's of HSA 96-NS(x:1) are plotted versus attachment ratio, x, in Figure 5 (open symbols) and compared with the behavior of HSA 99-NS(x:1) (filled symbols).At lower attachment ratios, HSA 96:NS(x:1) is actually less stable than HSA 99-NS(x:1) , indicating the proposed HSA 96-L-NS(x:1) structure (with the presence of FA-containing ligands) is less stable than (filled symbols).For HSA 96-NS(x:1) t m also approaches 75˚C at higher x values, but does so more slowly than HSA 99-NS(x:1) .
its HSA 99-NS(x:1) counterparts at the same values of x.At higher incubation ratios (≥1:20) t m 's of the thermograms level off to around 75˚C.
Figure 5 shows for HSA 96-NS(x:1) that t m also approaches 75˚C at higher x values, but does so more slowly than HSA 99-NS(X:1) .In values from Table 1 are plotted versus attachment ratio for the HSA 99-NS(x:1) and HSA 96-NS(x:1) preparations.This plot reveals differences at the lowest attachment ratios, the plots reaching near equivalence around x = 15.Interestingly, 0 37 G ∆ is greater for the HSA 96-NS(1:x) preparation, even though the t m is lower than that for HSA 99-NS(x:1) at comparable x.This observation could be due to effects of irreversible aggregation in the denaturation region.
HSA 99 (without the FA/G-LC contaminant) aggregates more readily in the denaturation region, i.e. has a higher rate of intermolecular aggregation mediated by the melted state than the populations of HSA 96-H and HSA 96-L that comprise the HSA 96 preparation [1].FA is known to mediate aggregation in the denaturation region, thus the greater stability of HSA 96-H .By the same argument, attachment of biotin to HSA 99 to form HSA 99-NS(1:x) must also act to decrease irreversible aggregation, as the stability of HSA 99-NS(1:x) is increased by more attachment.Then Figure 6 suggests irreversible aggregation in the denaturation region is increasingly discouraged by increased amounts of biotinylation.The result is values of for HSA 99-NS(x:1) that are smaller than for HSA 96-NS(x:1) at the lower attachment ratios (as plotted in Figure 6), but equivalent at higher ratios.
Prior to comparison, discussion and interpretation of tabulated thermodynamic parameters it should be noted that parameter evaluations were made under the assumption that HSA melting from the native to the denatured state predominantly occurs in a two-state manner.Several published studies have reported results of measurements on HSA and thermodynamic transition parameters of the HSA melting transition [1] [13] [17].In these reports different methods of data analysis, baseline treatment, model assumptions and curve fitting were employed.Ours is a standard approach, and arguably one of the least sophisticated that has been applied, with several simplifying assumptions.As discussed below relative comparisons of the parameters within the data set were considered to be quantitatively significant, and provided additional insight into the consequences of biotin attachment on the different HSA samples.Melting transition parameters evaluated by DSC analysis of HSA 99 , HSA 96 and their modified versions with biotin attached, HSA 99-M(x:1) , HSA 99-NS(x:1) , HSA 96-M(x:1) and HSA 96-NS(x:1) are summarized in Table 1.
A few general features of the parameter sets are noteworthy.1) For both preparations of unmodified HSA alone, HSA 99 and HSA 96 measured values ( This general agreement indicates our simplifying assumptions of a two-state melting transition with ΔC p = 0, and variations in baseline analysis procedures must have a small effect on the quantitative significance of the evaluated thermodynamic parameters.2) Response of the t m of HSA to covalent attachment of biotin is different for HSA 99 than for HSA 96 .For HSA 99-M(x:1) at (x = 1), the t m increases by over 5˚C, and does not change further at higher attachment ratios.The average (over all attachment ratios) is t m = 68.G ∆ for HSA 99-M(x:1) concurs with that for the t m .For HSA 99-M(x:1) 0 37 G ∆ increases to the same value at ratios of x = 1, 3 and 10 with an average value of 16.8 ± 0.9 kcal/mol.In contrast, under the same conditions for HSA 96-M(x:1) samples at attachment ratios of x = 1, 3 and 10 there is a nearly 13% decrease in G ∆ values for modified HSA 96-M(x:1) are identical to that of unmodified HSA 96 with average 0 37 G ∆ = 26.1 ± 0.9 kcal/mol.To summarize for the cases of HSA 99-M(x:1) and HSA 96-M(x:1) , both show no difference between the attachment ratios (x = 1, 3, 10), consistent with single site attachment.For HSA 99-M(x:1) biotin attachment results in an increase in stability ( 0 37
In contrast, opposite responses of G ∆ , to attachment of biotin to amines of lysine residues were found for HSA 99-NS(x:1) compared to HSA 96-NS(x:1) .For HSA 99-NS(x:1) the t m increases incrementally with increasing attachment ratios x > 20 for a total increase of nearly 10˚C, and levels off thereafter (see Figure 5).Examination of Table 1 indicates the thermodynamic parameters for HSA 99-NS(x:1) and HSA 96-NS(x:1) tend in opposite directions.In comparison consider the behavior of 0 37 G ∆ values versus attachment ratio as plotted in Figure 6.As shown there, for HSA 99-NS(x:1) at x = 1, 0 37 G ∆ increases and continues to do so in an incremental fashion up to an attachment ratio of x = 15 where after it levels off and is constant up to the highest ratios (x = 40, 50).In contrast for HSA 96-NS(x:1) , at an attachment ratio of x =1, 0 37 G ∆ decreases from the value for HSA 96 and decreases again for x = 3 and 5; dramatically increases at x = 8, then levels off at the higher ratios (see Figure 6).The opposite responses of the 0 37 G ∆ values for HSA 99-NS(x:1) and HSA 96-NS(x:1) as a function of increased attachment ratio reveals an effect of the presence of the small amount (3%) of FA/G-LC on modified HSA.The contrasting observations are: 1) Attachment increases stability ( ) of the HSA 99-NS(x:1) samples compared to HSA 99 alone.This stability enhancement occurs in an incremental fashion up to 1:15 were presumably saturation of available attachment sites is reached.2) Attachment dramatically decreases stability ( 0 37

G ∆
) of the HSA 96-NS(x:1) samples at the lowest attachment ratios (x = 1 -10); thereafter at increased attachment ratios (x ≥ 10) 0 37 G ∆ converges to a constant value of higher stability, but with less stability than HSA 96 alone.

Thermodynamic Interpretation of Results
In order to provide an interpretation of the thermodynamic measurements of the two forms of HSA and consequences of biotinylation of them, consider the following simple model of thermal denaturation of HSA written as the following reaction, 0 HSA HSA where the transition is assumed to occur in a pseudo two-state manner from the native intact structure, HSA N , to the denatured state, HSA D .This reaction occurs with an observed standard state equilibrium constant,  1) can be represented in terms of the corresponding chemical potentials as, where 0 N µ and 0 D µ are the hypothetical standard state chemical potentials for the native state (N) and the de- natured state (D), respectively.The standard state free energy, , is given in terms of the differences of the standard state chemical potentials of the native and denatured states, i.e.
The observed standard state equilibrium constant for the reaction is also given by, ( ) G ∆ values in Table 1.In terms of the specific samples at hand, for HSA 99 , 0 0 0 99 99 99 Δ 11.9 kcal mol Similarly for HSA 96 , If the denatured state for both species is arbitrarily assigned as the reference state, 0  ( ) with H x equal to the mole fraction of HSA 96-H in the HSA 96 preparation.Here it is assumed if a small amount of HSA 99 is present, it is so in a negligible amount (<5%).As such, writing Equation (8) in this manner ignores the slight presence of HSA 99 (reckoned as the small inflection on the low temperature side of the HSA 96 melting transition in Figure 1).Equation (8) assumes that other than HSA 96-H the remaining protein is in the HSA 96-L form.
As stated previously we assume HSA 96-L and HSA 96-H that comprise the HSA 96 preparation are in many ways analogous to the defatted (HSA L-F ) and un-defatted (HSA H-F ) forms of HSA previously studied and reported [1] [15] [16].Their different forms were observed under conditions very similar to ours (heating rate, buffer composition, protein concentration, relative fraction of FA).From their analysis they reported a value of x H in Equation (8) around 0.3 [1] [16].They also reported measured average values for the transition enthalpies for the HSA L-F and HSA H-F species.These were, ΔH L-F = 4.18 cal/g for the defatted species and ΔH = 5.16 cal/g, for the un-defatted species [1].The molecular weight of HSA (66,500 g/mol) provides ΔH L-F = 280.06kcal/mol and ΔH H-F = 345.72 kcal/mol.Reported t m values for HSA L-F and HSA H-F were 65.7˚C and 78.3˚C, respectively.These combined with ΔH L-F and ΔH H-F values provide an estimate on the transition entropies ΔS L-F and ΔS H-F for the reported defatted and un-defatted species.That is, 280060 826.5 cal K mol 338.85 G ∆ , of 23.9 kcal/mol and 39.6 kcal/mol were obtained for the HSA L-F and HSA H-F structures, respectively.In analogy with Equations ( 7) and ( 8) these In the discussion that follows it is arbitrarily assumed for the example shown that the values for HSA 96-L and HSA 96-H are approximately 75% of reported values for the presumably analogous forms, HSA L-F and HSA H-F .That is we assume − − − = kcal mol .This is probably not an unrealistic assumption based upon differences in data analysis and reported thermodynamic parameters compared to ours [1].For both sets of values the relative difference remains the same.
With these assumptions, estimates on the value of x in our experiments can be made by rearranging Equation ( 8), µ − above, but about 20% larger than reported [16] for the relative frac- tions of the analogous forms HSA L-F and HSA H-F .In the remaining development a value of 0.53 H x = is assumed for the initial composition of HSA 96 .

Attachment of Biotin to HSA
Consider the following cases.For the HSA 99 preparation, For the HSA 96 preparation the thermogram is comprised of two overlapping transitions.The lower temperature transition seems most affected by attachment and is attributed to HSA 96-L with biotin attached, i.e.HSA 96-L-M(x:1) , and assumed to be similar (but not identical) to HSA 99-M(x: 1 Rearranging Equation (10) and solving for 96 H M f − − provides an estimate on the fraction of HSA 96-H-M mole- cules that remain following the attachment reaction, i.e.
We assume that biotinylation of So the effect of biotinylation on the HSA 96 preparation is either to convert some fraction of the HSA 96-H-M molecules to the HSA 96-L-M form, or reduce the stability of HSA 96-H-M compared to HSA 96-H .
Results of the above analysis and behavior of the measured thermograms are consistent with the following scenario.In the process of the attachment reaction a portion of the HSA 96-H molecules get biotinylated and convert to HSA 96-L-M essentially becoming HSA 99-M , which is much less stable than HSA 96-H but more stable than HSA 99 alone.Thus, there are two opposing effects of biotin attachment on protein stability.Biotin attachment to the HSA 96-L and HSA 96-H structures to create HSA 96-L-M and HSA 96-H-M increases their stabilities.But with the attachment reaction a fraction of the more stable, HSA 96-H molecules are converted to the less stable HSA 96-L form, then subsequently biotinylated to become HSA 96-L-M ≈ HSA 99-M .In combination, the loss of a fraction of the HSA 96-H molecules through conversion results in a decrease in stability and reduces the chemical potential.This destabilization is overcome by attachment of biotin to HSA 96-L to create HSA 96-L-M resulting in an increase in chemical potential.This reduction and increase offset.As a consequence both the HSA 96-M and HSA 96 preparations have the same chemical potential.
Evidently, biotinylation increases stability of HSA 96-L-M while reducing stability of HSA 96-H-M compared to their unlabeled counterparts, HSA 96-L and HSA 96-H , respectively.It is possible that binding of ligands that comprise the FA/G-LC contributes to enhanced stability of HSA 96-H that must exist in a more stable conformation than HSA 96-L .HSA 96-L may bind to the FA/G-LC ligands and have a structure and stability like HSA 99 bound by the FA/G-LC ligands and have a different conformation than HSA 96-H .Clearly, the 3% contaminant, FA/G-LC, in the HSA 96 preparation has quite a profound effect on the structure and stability of the different forms of HSA that comprise the HSA 96 preparation.
For the case of multiple attachment, consider for the HSA 99 preparation,  1, and plotted versus attachment ratio, x, in Figure 6.Higher x results in increased stability of the HSA 99-NS(x:1) structure compared to HSA 99 .For the HSA 99 preparation the effect of biotinylation is to incrementally increase 0 37 G ∆ (and t m ) with increased attachment ratio, x.These results for HSA 99-NS(x:1) provide the basis for dissecting the effects of multiple attachment on the HSA 96 preparation.
On the HSA 96-NS(x:1) thermograms in Figure 4 there is no sign of the high temperature transition attributed to the aforementioned HSA 96-H structure.Apparently this would indicate an effect of the attachment reaction is conversion of all molecules to the HSA 96-L-NS(x:1) type structure.Based on this observation, we assume the thermograms of the HSA 96-NS(x:1) samples are comprised of contributions of the labeled HSA 96-L-NS(x:1) which is similar, but not identical (especially at low attachment ratios) to HSA 99-NS(x:1) ; and contributions from binding of the FA/G-LC to that structure, i.e. rapidly decreases to essentially zero at the higher biotinylation ratios.This decrease presumably corresponds to occlusion of FA/G-LC binding to HSA 96-L-NS(x:1) by increased biotin attachment at the higher ratios.Given the above analysis and interpretation derived therefrom, we can provide an explanation of the apparently contrasting behaviors of the HSA 99-NS(1:x) and HSA 96-NS(1:x) species.Consider the following scenario for HSA 96-NS(1:x) .At the lowest attachment ratios, the protein resides as HSA 96-L with biotin attached to a few sites and some FA/G-LC bound to other potential binding sites.This results in a structure with a stabilizing component in the biotin label (as indicated for HSA 99-NS(x:1) ) and a further stabilizing component due to binding of FA/G-LC to HSA 96-L-NS(x:1) .As the attachment ratio increases more biotin is attached, displacing formerly bound FA/G-LC at new biotinylated attachment sites, and occluding further FA/G-LC-binding there.At higher levels of biotinylation, destabilization brought about by the loss of FA/G-LC binding is more than compensated for by the increase in stability afforded by the increased amount of attached biotin, resulting in a more stable structure with increased t m and greater 0 37 G ∆ that approaches that of HSA 99-NS(x:1) for x > 20.Thus, the behavior of HSA 96-NS(x:1) can be summarized as follows.The attachment reaction results in conversion of the HSA 96-H species to HSA 96-L .At the lowest attachment ratios (x < 20), the HSA 96-L-NS(x:1) structure is stabilized by FA/G-LC binding and attachment of biotin.At higher attachment ratios stability of the biotinylated structure increases, but FA/G-LC is released resulting in a conversion of the structure from HSA 96-L-NS(x:1) to one more like HSA 99-NS(x:1) .If this is the case, the difference in the 0 37 G ∆ values for HSA 96-NS(x:1) and HSA 99-NS(x:1) in Equation ( 11),

Reversibility to HSA Thermograms
As mentioned previously, published melting studies of HSA reported observations, at temperatures approaching t m , of a second reaction attributed to irreversible aggregation of the protein in the denaturation region [1] [12] [13] [15].They noted the potential existence of a slow irreversible denaturation step(s) but concluded potential contributions to the overall measured thermogram were minimal.Irreversibility occurs due to intermolecular association of denatured HSA polypeptide strands.Such aggregation is not readily reversible in the transition region and can affect reversibility of the HSA melting transition [16].Influence of irreversible denaturation on the primary melting transition of HSA can be minimized (somewhat) through appropriate choices of experimental parameters [12] [13] [16].In effect, under optimally designed experimental conditions the temperature scanning rate is such that kinetics of the irreversible denaturation reaction are relatively much slower, and only begin to contribute significantly to the HSA melting transition at temperatures above the t m .Since our experiments of HSA were carried out under similar conditions to those previously reported [1], it was assumed the measured HSA melting transitions are, for the most part, reversible.Inherent in the analyses that were performed was the choice of the "universal" reference state, i.e. the denatured state.Therefore, the possibility of heterogeneous denatured states, differing in character in addition to their ability to reversibly renature from the denatured state to the native state, is not considered.
Contributions of the irreversible transition to the measured ΔC p values can also manifest in the thermogram baseline and therefore be reduced or subtracted out (somewhat) in the baseline analytical procedure.The irreversible reaction could also manifest in evaluated thermodynamic parameters of the melting transition, which assumes a truly equilibrium (reversible) process with parameters evaluated from the area under the baseline corrected thermogram.This process might influence absolute values of thermodynamic transition parameters, ΔH cal and ΔS cal , which may be lower (or higher) than what has been reported previously, depending on how much of the baseline encroaches on the melting of transition of HSA from the native to denatured form, and how much irreversibility affects the transition and choices made as part of the baseline analysis procedure.In our analysis as much as 30% of the total endotherm of HSA can be absorbed in the baseline.Our baseline analysis treatment was performed in a systematic fashion in precisely the same manner for all thermograms.Within the inherent uncertainties underlying the operative analysis, relative values of the thermodynamic parameters are considered to be more quantitatively significant.Fortunately the analysis that was performed involved relative comparisons of the transition thermodynamic parameters.

Conclusion
In summary, results of this study have demonstrated that DSC is a sensitive method for monitoring solution conformation and stability of proteins (HSA).These experimental results and model analysis indicate that HSA can exist in very different thermodynamic states depending on the presence of a very small fraction (3%) of contaminants (likely) containing FA and globulin, termed FA/G-LC.These different states of HSA are also differentially affected by biotinylation.Thus, both biotinylation (adduct formation) and binding of ligands that comprise the FA/G-LC can stabilize the native form of HSA, and the different forms of HSA differentially respond to biotinylation in the presence or absence of FA/G-LC ligands.

p
Consolidation of the thermogram inFigure 2(b) seems to arise from a shift up in temperature of the major peak attributed to labeled HSA 96-L (HSA 96-L-M(1:1)

Figure 5 versus x in Figure 6 ( 6
the t m 's of the HSA 99-NS(1:x) preparation (at least for x ≤ 20) indicate it is apparently more stable against thermal denaturation than HSA 96-L-NS(1:x) at the same x.The thermodynamic parameters evaluated for HSA 96:NS(x:1) and HSA 99:NS(x:1) ( not shown).For comparisons that follow values of t m and 0 37 G ∆ versus x are utilized.Observed trends in t m and 0 37 G ∆ with covalent attachment of biotin to HSA 99 and HSA 96 provide an indication of the effects of biotinylation on thermodynamic stability.As shown in Figure 5 and Figure 6, these parameters show very different responses of the two HSA forms to covalent modification by biotin.In Figure
7 ± 0.4˚C.Response of the ther- native structure is favored.The equilibrium reaction in Equation ( at 37˚C for both HSA 99 and HSA 96 are given as their 0 37 above requires slight modification to properly consider the HSA 96 preparation which, as stated earlier and evidenced by Figure1, is primarily comprised of two different populations of the protein, termed HSA 96-H and HSA 96-L .To account for this mixture we write, for the defatted and un-defatted forms of HSA.The difference, graphical analysis of the HSA 96 thermogram (not shown), it was assumed the composite curve in Figure1collected for the HSA 96 sample was comprised of just two major components, HSA 96-H and HSA 96-L .The graphical curve analysis returned an estimate of 0.5 H x = which is in agreement with what was calculated assuming the values of 0 96 H µ − and 0 96 L − represents the generic attachment reaction that depends on the site of attachment, Bio = M or NS.Consider single site attachment first, i.e.Bio = M.In analogy with Equation (6) the observed standard state free-energy of the melting reaction for HSA 99-M is given by, the attached biotin increases the chemical potential of the HSA 99-M(x:1) structure compared to HSA 99 .In the above we have used the average value evaluated from the average over the values obtained for the HSA 99-M(x:1) samples at x = 1, 3, and 10.
the same effect as biotinylation of 99 HSA , and similarly write the analogous chemical potentials, i.e.This indicates that the fraction of molecules in the HSA 96-H-M form decreases from 0.53 to 0.24 when the single site labeling reaction takes place.The result is approximately 50% of the HSA 96-H in the original HSA 96 preparation being converted in the single site attachment reactions.Alternatively, consider the fraction of molecules 96 H M f − − in the HSA 96-H-M state (presumably now labeled) does not change during the attachment reaction, i.e.
attachment ratio is a direct gauge of the added stabilization afforded by FA/G-LC binding to the HSA 96-L ≈ HSA 99 structure.

Figure 7 .
Figure 7. Values of the additional contribution to the chemical potential of HSA due to FA/G-LC binding, ( ) 0 0 96 :1 FA FA NS x µ δµ − − = (see text) plotted versus attachment ratio, x.

Table 1 .
Thermodynamic transition parameters.Values for the standard state enthalpy temperature, t m , measured and evaluated in DSC experiments for the samples of HSA that were prepared.Values are considered accurate to within 5%.Each transition temperature, t m , is an average value that does not vary more than 0.5˚C in multiple experiments.

Table 1 .
The evaluated parameters, t m ,

Table 1 .
Comparison of the evaluated thermodynamic parameters in Table It is possible that during the attachment reaction some of the labeled HSA 96-H molecules (HSA 96-H-M ) convert to the HSA 96-L-M form.To account for these considerations the measured chemical potential of the HSA 96-M preparation is written as,