Transitions of serum albumin in patients with glomerulosclerosis ‘in vivo’ characterization by...

Maurizio Bruschi1

Luca Musante1

Giovanni Candiano2

Laura Santucci1

Cristina Zennaro1, 3

Michele Carraro3

Piero Del Boccio4, 5

Rosanna Gusmano1

Francesco Perfumo2, 6

Andrea Urbani4, 5

Gian Marco Ghiggeri2, 6

1KIDneyFOUNDation for RenalResearch in Children,Genoa, Italy

2Laboratory on Pathophysiology ofUremia, G. Gaslini Children Hospital,Genoa, Italy

3Cattedra di Medicina Clinica,University of Trieste, Italy

4Department of Biomedical Science,Università degli Studi di Chietie Pescara, Italy

5Centro Studi sull’Invecchiamento,Fondazione Università“G. D’Annunzio”,Chieti, Italy

6Department of Nephrology,G. Gaslini Children Hospital,Genoa, Italy

Received August 31, 2005Revised December 16, 2005Accepted January 2, 2006

Research Article

Transitions of serum albumin in patients withglomerulosclerosis ‘in vivo’ characterizationby electrophoretic titration curves

HSA functions as a physiological transporter of solutes and small molecules thatinduce structural transitions ‘in vitro’. Analysis of these transitions requires prior purifi-cation of HSA that could introduce bias due to conformational changes. We utilizedelectrophoretic titration curves to describe a neutral to acid (N–A) transition of HSAdirectly in sera of seven patients with active focal segmental glomerulosclerosis(FSGS). The divergent electrophoretic profile of HSA was characterized by a shift in therange of pHs between 4.5 and 7.5 with an average variation of free electrophoreticmobility corresponding to loss of 1 positive charge in the pKa protonation range ofhistidyl residues and should involve domain I of HSA. ‘In-gel’ determination by mal-eimide-PEO2-biotin of free SH 34 of domain I showed inaccessibility of the dye at thissite in pathological HSA and alkylation with the same complex induced N–A transitionin normal HSA. Potential binders of free imidazoles such as Ca11 and/or of SH 34 suchas NO were excluded on the basis of direct titration and studies on binding stimulation.This is the first report describing a transition of HSA directly ‘in vivo’, and the utilizationof electrophoretic titration curves was critical to this purpose. This transition appearsto be specific to FSGS and is unrelated to the nephrotic syndrome, Ca11 and NObinding. Spectroscopic analysis will elucidate the structural implication.

Keywords: Albumin / Electrophoretic titration curves / Focal segmental glomerulo-sclerosis / Nephrotic syndrome DOI 10.1002/elps.200500641

1 Introduction

HSA, the major circulating protein, is implicated in anarray of physiological functions [1–10] varying from main-tenance of plasma oncotic pressure and regulation ofvascular tone by NO [1–3] to transport of hormones, bio-logical substances, heavy metals and ions [4–6]. Finally,HSA also exerts a major antioxidant function againstreactive oxygen species that could play relevant rolesunder pathological conditions [8–10]. Probably the listlacks completeness and studies in progress will defineunexpected details.

Most of the reasons for this amplified functionalpanel resideonthe plasticityofalbumin thatmayachieve conformationalchanges or transition in relation to pH and/or bindingmicroenvironments that modify the pKa of ionized groups[11–13]. The basic molecular configuration of HSA asdetermined by X-ray crystallography includes three-struc-tural homologue domains (domains I, II and III) each formedby two subdomains in which are located hydrophobicbinding sites [14, 15]. Three-dimensional structure is givenby disulphide pairings among the 17 cysteines of thesequence which form eight disulphide bonds with a sul-phydryl group inposition 34 left free to reactwith thiolsof theenvironment. Domains II and III are the site of most bindingsincluding fatty acids, bilirubin, chemical and drugs. Fewcompounds react at domain I, majorexceptions being Ca21

[12, 16], that interacts with histidyl residues and NO thatreacts with the free SH residue in position 34 [1].

In the pH range from 7 to 8.5, HSA undergoes the so-called N–B transition that is mainly due to deprotonationof histidyl residues and involves conformational changes

Correspondence: Dr. Gian Marco Ghiggeri, Laboratory on Patho-physiology of Uremia, G. Gaslini Children Hospital, Largo G. Gaslini,5. 16148 Genova, ItalyE-mail: [email protected]: 139-010-395214

Abbreviations: FSGS, focal segmental glomerulosclerosis; PAN,puromycin aminonucleoside

2960 Electrophoresis 2006, 27, 2960–2969

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2006, 27, 2960–2969 Proteomics and 2-DE 2961

with disruption of salt bridges between domain I and thecarboxyl-terminal of domain III. Bindings to domain I ofHSA such as in the case of Ca21 ions determine, instead,N–A transition that results from a downward pKa shift ofhistidyl residues [12]. All studies describing transitionstates of HSA have been performed ‘in vitro’ but a moredetailed analysis in humans is warranted. One possibleapproach is the purification of albumin by precipitation inpolar environments and/or exposition to high osmoticmedium that modify the basic conformation of the proteinand prevent spectroscopy analysis. It is also expectedthat purification steps commonly utilized for HSA such asprecipitation and/or pseudo-ligand chromatographyshould weaken and lose transported ligands which is oneof the key aspects of albumin transition. This problemaccounts for the lack of data on ‘in vivo’ HSA transitionunder normal and disease conditions.

We have utilized electrophoretic titration curves to ana-lyse HSA in patients with focal segmental glomerulo-sclerosis (FSGS) and nephrotic syndrome that is a majorrenal disease causing loss of albumin in urine. This is a2-D electrophoretic technique in which proteins moveperpendicular to a pH gradient generated by a stack ofcarrier ampholytes and generate sigmoidal pH/mobilitycurves that are proportional to chemical titrations. It wasfirst developed for single proteins by Rosengren et al. [17]and then extended to the analysis of mutant phenotypesand protein–ligand interaction by Righetti et al. [18] whofirst proposed the term of electrophoretic titration curve.Attanasio et al. [19] finally demonstrated good corre-spondence of calorimetric and pH/mobility curves of sin-gle proteins utilizing the Linderström–Lang (Eq. 1) [20] tocalculate number and mean pKas of any given ionizationgroup and defined the mathematical parameters ‘z’ netcharge, ‘w‘ electrostatic factor and the frictional coeffi-cient ‘f’ for defining the number of ionized groups at anygiven pH. The great advantage of electrophoretic titrationis that it allows a direct evaluation of protein charges in apH gradient between 4 and 9 without the need of pre-purification of the protein that appears to be particularlysuitable to analyse HSA transition.

2 Materials and methods

2.1 Chemicals

Ampholines were from Amersham Bioscience (Uppsala,Sweden); acrylamide N,N 0-methylene-bisacrylamide(Bis), SDS and N,N,N 0,N 0-tetramethylethylenediamine(TEMED) electrophoresis calibration standard were fromBioRad Laboratories (Hercules, CA, USA); puromycinaminonucleoside (PAN) was obtained from Sigma (St.

Louis, MI, USA); maleimide PEO2-biotin (biotinyl-3-maleimidopropionamidyl-3,6-dioxactanediamine) wasobtained from Pierce (Rockford, IL, USA). All otherchemicals, of analytical and electrophoretic grade, werepurchased from BDH (Poole, UK). Solutions were pre-pared fresh using Milli-Q water.

2.2 Patients

Sera were obtained from seven patients with FSGS andflorid proteinuria with nephrotic syndrome. Patients withproteinuriareceived treatment with steroids alone (at vari-able doses) or in association with cyclosporin, ACE in-hibitors or statins, according to consolidated schemes(ISKDC). Cyclosporin was given at 5 mg/kg starting dose,followed by tapering to reach the minimum dose requiredfor maintaining cyclosporin serum levels between 50 and100 ng/ml. Four patients of the former group were alsoevaluated after 1 year of stable remission induced bydrugs. The control group consisted in ten normal donorsof the lab staff or their sons (five female, five male, meanage 18 years, range 10–34). General clinical parametersincluded blood cell counts, serum creatinine and urealevels, cholesterol, triglycerides and liver function testsperformed according to international standardized pro-cedures (Table 1).

Adult patients and parents (for patients under 18 years)were requested to give informed consent for DNA analy-sis (a part of the clinical diagnosis of FSGS) and forreviewing their clinical parameters on a statistical basis.

2.3 In vivo modification of HSA transition byhyperventilation

In vivo modification of HSA transition were achieved byinducing respiratory alkalosis in two normal volunteers(males, 52 and 43 years) who practised forced respirationfor 10 min in a normal atmosphere. Blood samples weretaken from a peripheral vein at baseline and after 10 minhyperventilation that induced changes of vein pH/pCO2/HCO3, ionized Ca11 (Table 2) while maintaining stablelevels of serum albumin and total Ca11.

2.4 Experimental nephrosis in rats

Sprague-Dawley/CD male rats (Charles River, Calco,Italy) weighting 200 to 250 g at the start of the experimentwere used. Nephrosis was induced by a single injection ofPAN, 7.5 mg/100 g in normal saline through the tail vein ofnonanesthetized animals [21]. Control rats were givennormal saline. Proteinuria developed in all treated rats


2962 M. Bruschi et al. Electrophoresis 2006, 27, 2960–2969

Table 1. Clinical data of seven patients with proven FSGS based on clinical and histology findings.Four patients (Pt 4, 5, 6,7) were evaluated during the florid phase of nephrotic syndrome andafter stable remission induced by drugs. Two were still receiving cyclosporin at the time ofre-evaluation

Age atobservation

Age atonset(years)

ProtU

gr/24 hSerumcreatinine(mg%)

Serumcholesterol(mg%)

Therapy Histology

FSGS-Proteinuric

Pt 1 14 6 2.5 0.7 344 CsA FSGSPt 2 5 5.2 2.7 2.6 290 St/Ace i FSGSPt 3 10 5 2 1.1 194 St/CsA/Ace i FSGSPt 4 5 3 2.7 0.4 453 CsA FSGSPt 5 2 1 6.6 0.9 220 CsA/St/Statin FSGSPt 6 10 2 4 1.1 240 St/CsA/Ace i FSGSPt 7 13 7 2 0.8 190 CsA FSGS

FSGS-Remission

Pt4 9 3 0.1 0.5 150 2 FSGSPt5 10 1 0.3 120 180 2 FSGSPt6 20 2 0.3 0.8 155 CsA FSGSPt7 21 7 0.3 0.9 160 CsA FSGS

Abbreviations: CsA, cyclosporin; St, steroids; Ace i, Ace inhibitor; FSGS, focal segmental glomer-ulosclerosis.

Table 2. Ionization parameters of normal and pathologi-cal HSA calculated according to Linderström–Lang Eq. (1). In bold it is reported that patho-logical HSA presents only one imidazole residuein respect to normal HSA

Groups Normal HSA Pathological HSA

n pKa n pKa

a-COOH 1 3.8 6 0.2 1 3.8 6 0.3r-COOH 96 4.00 6 0.02 96 4.00 6 0.03Thiolic 1 8.0 6 0.3 1 8.0 6 0.2Phenolic 18 10.2 6 0.2 18 10.2 6 0.3Imidazole 2 6.24 6 0.01 1 6.23 6 0.01a-Amino 1 7.80 6 0.02 1 7.80 6 0.02e-Amino 59 10.4 6 0.4 59 10.4 6 0.5Guanidyl 24 .12 24 .12

pI 4.79 6 0.01 4.68 6 0.01

after a mean time of 15 days from PAN loading; bloodsample were withdrawn from the tail vein after an ade-quate period of stable proteinuria.

2.5 Electrophoretic titration curves

Electrophoretic titration curves were done utilizing serumand, in some cases, plasma in ACD. The general meth-odology was performed according to Righetti [18].

Square gel slabs (145 mm6145 mm61.5 mm) were castto contain acrylamide (%T 5.00,%C 2.67) and 2.5% w/vcarrier ampholytes, pH 3.5 6 10. The first-dimensionalseparation was run at 100 V for 5 min, 200 V for 30 min,300 V for 30 min and then 400 V for 30 min, until the pHgradient was fully developed and the system reached asteady state. After that electrode gel layers were cutaway, gels were turned 907 and the trench was filled withserum (200mg of protein). After new electrode strips hadbeen overlaid onto the gel electrophoresis was performedperpendicular to the first dimension at 200, 300 and 400 Vfor 5, 10 and 30 min, respectively, at 137C. Electrolyteswere 0.25 M NaOH at the cathode and 0.25 M H3PO4 atthe anode. At the end of the experiment, the pH gradientwas measured into 22 gel slices, each 0.5 cm wide, per-pendicular to the central trough; each slice was elutedwith 5 mL of 10 mM KCl and read with a digital pH meter.Equations, calculations of electric mobility and validationof electric titration curves for globular proteins are report-ed in our previous paper [18, 19]. Net charge estimation,Z, was calculated according to Linderström–Lang (Eq. 1)with a pH range between 2 and 10, assuming that (i) theprotein remains compact and impenetrable to solventstructure, (ii) the protonable groups are all exposed to thesolvent and (iii) no structural changes occur throughoutthe pH range.

pH� logni

n0i � ni

!¼ pK0

i �1

2:3RT

� �wZ (1)



Considering both number and pKas of each ionizationgroup, Eq. (1) can be developed into Eq. (2) that allowssimultaneous analysis of all variables.

pH�X

Logni

n0i � ni

!¼X

pK0i �

12:3RT

� �wZ (2)

Electrophoretic mobility values are related to the netmacromolecule charge, Z, calculated from Eq. (1),according to the following Eq. (2)

U ¼ vE¼ Ze

f(3)

where U is the electrophoretic mobility, v is the migrationvelocity, E is the electric field strength, f is the frictionalcoefficient and e is the electron elementary charge.

Staining of proteins was done by Coomassie R-250 withsome modifications as described in [19, 22]. Stained gelswere digitized by using a GS 800 scanner (BioRad) andanalysed by QuantityOne program (BioRad) that gives adirect merge of two curves after false labelling to under-line differences. According to this technique in severalexperiments HSA was labelled in red (FSGS, hyperventi-lation, FSGS 1 maleimide, PAN-rats) while it was in greenfor each normal control. The mobility was calculated asmigration distance divided by the product of migrationtime (s) and field strength (V/m).

2.6 Alkylation by maleimide and bymaleimide-PEO2-biotin

The free SH group 34 endowed in domain I of normal HSAwas modified by 2 mM maleimide alone in PBS pH 6 for1 h at room temperature in the dark and by the maleimidePEO2-biotin (biotinyl-3-maleimidopropionamidyl-3,6-dioxactanediamine) under the same conditions. Alkyla-tion after maleimide treatment was checked by maleimi-de PEO2-biotin (biotinyl-3-maleimidopropionamidyl-3,6-dioxactanediamine) according to Pierce manufacturerinstructions. In this case, after alkylation maleimide wasremoved by precipitation in acetone. Serum proteins werefirst separated in mono-dimensional polyacrylamide gelsperformed according to Laemmli [22] with and withoutprior reduction with DDT.

2.7 In-gel determination of free SH residue 34

Free SH group reactivity of HSA were determined with themaleimide-PEO2-biotin (biotinyl-3-maleimidopropionami-dyl-3,6-dioxactanediamine) assay (Pierce) according tothe manufacturer instructions. After reaction with thecomplex maleimide-PEO2 proteins were precipitated withcold acetone for 20 min at 2207C and centrifugation at

10 0006g for 20 min. Proteins were then resuspended inPBS and a part was reduced by adding 30 mM DTE inLaemmli sample buffer. Separation was done with mono-dimensional in polyacrylamide gradient (T = 8–16%C = 2.6%) [22]. After electrophoresis, proteins weretransferred to NC and detected with neutravidin con-jugated with horseradish peroxidase. Biotin incorporationwas determined with (2-(4’-hydroxyazobenzene)-benzoicacid) utilizing the EZ™ Biotin Quantitation Kit (Pierce) at500 nm with correction for the amount of albumin asdetermined by Coomassie R-250.

2.8 In-gel determination of nitrosylation

Nitrosylation of HSA (30 mg load) was indirectly evaluatedin-gel after prior alkylation of free SH groups by methyl-methanethiosulphonate (MMTS). MMTS excess waseliminated with precipitation of proteins in cold acetoneas above and then denitrosylation was achieved byascorbate (NitroGlo™Nitrosylation Detection Kit, PerkinElmer, Boston, MA, USA). Ascorbate (2.5 mM final con-centration) was added before electrophoresis with 4 mMHPDP-biotin (biotin maleimide) and allowed to stand atroom temperature for 1 h. At this point the biotin mal-eimide-HSA complex was separated from other serumproteins by mono-dimensional electrophoresis as aboveand the presence of biotin linked to HSA was shown withneutravidin as for SH determination (see above). A posi-tive control for the formation of an NO-protein adduct wascreatine kinase (5 mg load) that was nitrosylated in vitrowith sodium nitroprusside (SNP) as described by Jaffrey[23].

3 Results

3.1 N–A transition of HSA in FSGS patients isrevealed by electrophoretic titration curves

Sera from a group of patients with FSGS were analysedby electrophoretic titration curves and compared to nor-mal serum. Since HSA accounts for almost 50% of allproteins, it emerges as a single major band with pI of 4.5–4.7 among other minor components and the electropho-retic behaviour can be easily described on this basis; HSAidentity was also confirmed by PMF. In Fig. 1a, the elec-trophoretic pattern of a sample of HSA from the FSGSstudy group, stained with Coomassie, is reported, whileFig. 1b reports the overlay of the same sample (red) withnormal serum (green) as digitalized and analysed bydirect image analysis. Titration curve of HSA calculatedfrom the electrophoretic migration at different pH values isshown in Fig. 1c, which shows the best fitting of patho-



Figure 1. Electrophoretic titration curve of a serum sam-ple from a patient with FSGS and for comparison of nor-mal serum. a) Serum from FSGS stained with CoomassieR-250; b) merging of the same serum as before (red) withnormal serum (green); c) titration curve of HSA calculatedfrom the electrophoretic migration at different pH values.Continuous and dotted lines represent the best fitting ofpathological and normal HAS, respectively, of calculatedvalues obtained from Eqs. (1) and (2) using ionization pa-rameters reported in Table 2. Solid circles show experi-mental migration values of pathological HSA; open uptriangles show normal HSA.

logical and normal HAS, respectively, deriving from cal-culated values obtained from Eqs. (1) and (2) using ioni-zation parameters reported in Table 2. This table reportsthe mean 6 SD of ionization parameters that reproducedexperimental curves of ten normal controls and seven

patients. In Fig. 1c, solid circles that represent experi-mental migration values of pathological HSA and con-tinuous line that represents the calculated curve overlay.The same occurs for normal HSA whose experimentalcurve is represented, in the same figure, by open triangleswhile the calculated curve is the dotted line. A divergenceof the electrophoretic profile of HSA from normal peopleand patients with FSGS, in the range of pH between 4.5and 7.5, is evident. FSGS sample migrates, in fact, fasterthan normal HSA, while above and below this range thetwo samples form a single yellow curve with completecorrespondence of electrophoretic behaviours. This pat-tern was constant in all patients presenting florid protei-nuria while a normal profile was restored after remissionof proteinuria in three patients receiving cyclosporin.According to Linderström–Lang equation Eqs. (1) and (2)and considering parameters reported in Table 2, this dif-ference can be attributed to the lack of a positivelycharged group with pKa = 6.24 corresponding to an imi-dazole residue. This change in pathological HSA is highlyreminiscent of the so-called N–A transition that resultsfrom perturbation of domain I of HSA due to binding ofCa11 in vitro.

3.2 Free SH group 34 alkylation and exclusion ofNO-HSA adducts

To address a specific implication of the unique free SHgroup 34 in domain I of HSA the following experimentswere performed: (i) in-gel determination of free SH residue34 in normal and pathological HSA (Fig. 2); (ii) titrationcurves of normal HSA after alkylation with maleimidealone or with maleimide-PEO2-biotin (Fig. 3a–c). Resultsfrom direct determination of free SH demonstrated nosignal from the complex maleimide-PEO2-biotin-neu-travidin in case of pathological HSA and positive signalfrom normal HSA under nonreducing conditions (seearrow in Fig. 2a). Both samples analysed under reducingconditions gave positive signals for the presence of freeSH residues (see arrow in Fig. 2b). Since, in the assay,reduction of HSA takes place only after the labelling withmaleimide, absence of signals in the former case cannotbe explained by lack of free SH but better indicates inac-cessibility of neutravidin due to steric hindrance in do-main I that contains SH 34. The following results fromalkylation of normal HSA confirm this possibility: (i) alky-lation by simple maleimide (Fig. 3a) did not modify theelectrophoretic profile; (ii) alkylation by maleimide-PEO2-biotin induced the N–A transition (Fig. 3b). Comparison ofthe two curves shows that for normal HSA treated withmaleimide alone (open triangles) the calculated curve(dotted line) coincides with the experimental curve. Whennormal HSA was pretreated with maleimide-PEO2-biotin



Figure 2. In-gel determination of free SH 34 by mal-eimide-PEO2-biotin. FSGS (track 1) and normal (track 2)sera were analysed for the presence of free SH, without(a) and with (b) DTE treatment. Arrows indicate albuminthat gives a negative image in absence of reduction.

(solid squares), transition was reproduced (Fig. 3c). Thisobservation indicates that N–A transition is reproduced byinsertion at domain I of the PEO2-biotin-neutravidin com-plex. Since one of the putative substance that commonlybinds HSA at the free SH residue 34 in vivo is nitric oxide [1,2], further experiments were undertaken to demonstrateSH nitrosylation utilizing an in-gel technique that inducesdenitrosylation by ascorbic acid (Fig. 4). This techniquefailed to demonstrate formation of NO-SH 34 adducts inboth normal (track 3) and pathological (track 4) HSAwhile apositive internal standard (creatine-kinase) was foundnitrosylated after SNP treatment (track 2) but not before(track 1). In the assay the free SH 34 after denitrosylation isevaluated, that is the binding site of NO, with the sameneutravidin assay utilized in the PEO2 assay that gives veryclear staining at the same protein level. This excludes lackof sensitivity of the detection assay.

3.3 Electrophoretic titration curve of HSA innephrotic rats

Electrophoretic profile of albumin was also evaluated inrats with experimental PAN nephrosis (see Section 2) thatis considered a good model of experimental nephrotic

Figure 3. a) Overlay of normal HSA alkylated (red) andnonalkylated (green) with maleimide; b) same samples asabove alkylated with maleimide-PEO2-biotin; c) titrationcurve of HSA calculated from the electrophoretic migra-tion at different pH values. Dotted lines represent the bestfitting of normal HSA of calculated values obtained fromEq. (1) using ionization parameters reported in Table 2.Open up triangles show experimental migration valuesafter alkylation of normal HSA with maleimide alone; solidsquares indicate normal HSA after alkylation with mal-eimide-PEO2-biotin.

syndrome[21]. HSA from PAN rats with massive protei-nuria (red curve) and from pair-age normal rats (greencurve) are visible as a single yellow curve, which indicatescomplete overlap of the two proteins (Fig. 5). This resultsuggests that nephrotic syndrome ‘per se’ is not a deter-minant of the change of electrophoretic characteristicsobserved in human FSGS.



Figure 4. In-gel analysis of NO-protein adducts. Tracks 1and 2 show creatine-kinase prior and after SNP treatmentthat induces marked nitrosylation. Tracks 3 and 4 shownormal and pathological sera, respectively. Same amountof sera utilized for in-gel determination of free SH wasloaded and free SH 34 was detected, after denitrosyla-tion, with the same neutravidin technique; this rules out asensitivity problem. By this assay we failed to detect sig-nificant nitrosylation of HSA.

3.4 Electrophoretic titration curves of HSAduring hyperventilation

A final putative modifier of HSA transition is Ca11 thatbinds the protein at domain I. An interesting way toachieve Ca11 binding to HSA is to determine a decrease

of H1 by respiratory alkalosis through hyperventilation.Table 3 shows clinical parameters at baseline and at theend of hyperventilation performed for 10 min by two nor-mal young volunteers that developed respiratory alkalosiswith 50% fall of pCO2 from 55 to 25 mmHg and a variationof pH from 7.32 to 7.46 with mild compensatory decreaseof blood HCO3. In parallel, levels of ionized Ca11

decreased from 2.32 to 0.7 with constant amount of totalCa11 that means a net 3.2-fold increment of the quotatransported by HSA. Electrophoretic titration curves ofserum before (green) and after hyperventilation (red) arereported in Fig. 6a, which shows a shift of the curve in thepH range between 4.3 and 6.0. Comparison of the twocurves is reported in Fig. 6b in which dotted lines repre-sent the best fitting of normal HSA and open squaresshow experimental migration values of normal HSAbefore hyperventilation; solid triangles indicate normalHSA after hyperventilation. The peak profile of Ca11

induced transition is therefore different from the one ofHSA in FSGS. Further experiments done with albumintreated with ACD, which is a strong Ca11 chelator, indi-cate the persistence of transition and exclude an impli-cation of Ca11 binding (not shown).

4 Discussion

FSGS is a chronic glomerular disease characterized bymassive loss of albumin in urine and hypoalbuminemiathat requires albumin reinfusion. These clinical elementsstimulated, in the past, some research on HSA in FSGSthat was limited to the interaction with fatty acids inhumans and in animal models [24, 25] but unfortunatelythe results produced only limited rebounds in clinicalpractice. The interest in HSA in FSGS has been renewedby recent findings on potential permeability effects ofHSA in isolated glomeruli [26, 27], which have been par-alleled by significant advancements in the knowledge ofphysiological functions of this protein. Among others, thetransport and release of substances active on vessels,such as NO, and antioxidant functions [1, 10] attractedour interest since they are of putative implication in FSGS.To accomplish these roles, HSA undergoes conforma-tional changes or transition in relation to pH changes thatmodify the pKas of ionized groups [11–13, 16]. Most of the

Table 3. Variation in clinical parameters before and after hyperventilation practised by two normal volunteers for 10 min

n v pH v PCO2

(mmHg)v HCO3

(mEq/L)Ca11 Total Ca

(mEq/L)Na Serum albumin

(gr%)

Baseline (2) 7.32 6 0.3 55 6 2 24 6 1 2.32 6 0.1 4.5 6 0.2 140 6 2 3.5 6 0.2Hyperventilation (2) 7.46 6 0.2 25.2 6 2 20.4 6 0.5 0.71 6 0.15 4.6 6 0.1 142 6 4 3.7 6 0.3



Figure 5. Electrophoretic titra-tion curves of rat albumin innormal rats (green) and afterinduction of nephrotic syndromeby PAN (red). Two curves mergeas a single yellow curve indicat-ing no changes induced bynephrotic syndrome.

data on HSA transitions derive from in vitro studies thatmimic to some extent the in vivo milieu but utilize stressedexperimental approaches which are not the same as theclinical conditions. A simplified scheme should considerat least two transitions also known as N–B (neutral tobasic) and N–A (neutral to acid) that are determined bydifferent mechanisms and involve separate domains ofthe protein. N–B transition involves conformational chan-ges and exposition of sulphydryl groups and makes theprotein more prone to release substances or interact withthe microenvironment. Overall the N–B transition plays arole in transport and release of drug/other molecules andin other key functional activities such as the sacrificialantioxidant effect of HSA. At the opposite, N–A transitioninvolves domain I of HSA and is mainly induced by bind-ing of Ca211 ions [12, 16, 28].

In this paper we describe an anomalous transition of HSAin a cohort of patients with FSGS and for this study wetook advantage of the high versatility of electrophoretictitration curves that allows a direct analysis of the proteinin-gels. This technique was first developed for optimizingpH conditions for electrophoresis and then extended tothe analysis of mutants, mixture of proteins and/or theirinteraction [18]. One major limitation of the technique isthat, with complex structures, it cannot allow directdetermination of any single ionization pKas which vary inrelation to conformational changes. On the other hand, agreat advantage of the technique is that it allows theanalysis of proteins with subtle electric and/or conforma-tional changes without the need of purification steps thatmay introduce potential structural bias. Attanasio et al.[19] compared calorimetric and electrophoretic titration ofCel 45 utilizing the Linderström–Lang equations (Eqs. 1

and 2) to determine both the number and mean value ofeach ionization pKas showing good correlation and sug-gested extension of electrophoretic titration to the analy-sis of other proteins. Starting from titration of mean pKasof ionization groups of bovine albumin reported by Tan-ford [20] and utilizing the combination of Linderström–Lang (Eqs. 1 and 2) and electrophoretic migration (Eq. 3)equation proposed by Attanasio [19] we calculated bothnumber and mean pKas that reproduce the experimentalcurve of normal and pathological HSA. These parametersare reported in Table 2 where SD indicates interindividualvariability. It was concluded that the divergence of theelectrophoretic curve in patients with active FSGS (vs.normal) in the pH range between 4.5 and 5.5 results fromloss of 1 positive charge in the pH range of ionization ofhistidyl residues. This electrophoretic pattern is stronglyreminiscent of N–A transition described above thatinvolves a modification of domain I of the protein. Ontheoretical basis, post-translational modification of func-tional groups at this domain cannot be excluded, since,well-known, imidazole groups can be readily modified bysugars and other reactants. However, experimentsaddressing the role of the free SH 34 strongly suggestedan implication of steric hindrance at domain I of HSA(alkylation by maleimide-PEO2-biotin) that cannot beexplained by chemical modification of imidazoles. Spec-troscopic analysis in an extended number of pathologicalHSA is now in progress to elucidate the molecular basisfor this phenomenon.

Waiting for structural answers, we tried to delineate theclinical boundaries of HSA transition and have extendedthe analysis to FSGS patients in remission from protei-nuria and to rats with nephrotic syndrome. It was



Figure 6. Electrophoretic titra-tion curve of (a) serum before(green) and after hyperventila-tion (red) as described inSection 2. b) Titration curve ofHSA calculated from the elec-trophoretic migration at differentpH values. Dotted lines repre-sent the best fitting of normalHSA of calculated valuesobtained from Eqs. (1) and (2)using ionization parametersreported in Table 2. Opensquares show experimentalmigration values of normal HSAbefore hyperventilation; soliddown triangles indicate normalHSA after hyperventilation.

demonstrated that transition in HSA is (i) specific forFSGS and (ii) unrelated to nephrotic syndrome per se andto drugs. Based on the bulk of data on HSA transitionsobtained in last decades by in vitro biochemical studieswe undertook a structural approach to define an implica-tion of recognized binders to domain I of the protein suchas NO and/or Ca11. Formation of HSA-NO adduct is ofparticular interest since it involves the binding of NO tothe same free SH 34 in domain I of the protein described

above. To determine the extent of nitrosylation an in-geltechnique based on titration of free SH groups was uti-lized after blocking free residue by biotin maleimide anddenitrosylation with ascorbate. We failed, however, toappreciate a significant amount of NO bound to HSAwhile other recognized carriers such as creatine kinasewere massively nitrosylated. Ca11 is the second putativebinder to domain I of HSA. To consider an effect of Ca11

on HSA transition an in vivo approach was utilized



according to which two normal volunteers developedrespiratory alkalosis trough hyperventilation that deter-mined a drastic 3.5-fold increase in Ca11 transported byHSA. In other words, hyperventilation determined amarked increase of Ca11 binding to HSA by decreasingfree H1 concentration in the blood due to respiratoryalkalosis. Analysis of HSA during hyperventilation showeda change of the titration curve with a major shift betweenpH 4.3 and 5 that mimics only in part the changeobserved in nephrotic patients. Pretreatment of HSA bystrong chelators such as ACD reverted the titration be-haviour of the hyperventilation states but did not modifyFSGS albumin. Therefore, results on HSA during hyper-ventilation exclude in a fairly conclusive way a relationwith Ca11 and are open to other unexplored possibilities.We must, in particular, consider substances that bindHSA at the site described above and should employspectroscopic techniques coupled with conformationalanalysis. The starting point is to verify by alternativetechnologies whether the absence of free SH residuesthat characterizes HSA in patients with FSGS is due tochemical linkage or could be better explained on thebasis of inaccessibility of the functional groove containingthe free SH.

In conclusion, we describe here a microtransition of HSAin patients with FSGS that involves domain I of the proteinwith loss of one histidyl residues. Indirect evidence sug-gests the presence of uncharacterized substance at thissite. This is the first description of an in vivo transition ofHSA and we must stress that utilization of electrophoretictitration curves was critical to demonstrate such a mod-ification. While two common binders to domain I of HSAsuch as NO and Ca11 could be excluded on an experi-mental basis, the definition of the mechanism remains anobjective for future studies. Extension of this approach toother pathological conditions is warranted.

This work was done with the financial support of the ItalianMinistry of Health and from of KIDney FUND. Authors alsoacknowledge Fondazione Mara Wilma e Bianca Querci forthe financial support to the project ‘Nuove evoluzioni sullamultifattorialità della sindrome nefrosica’.

5 References

[1] Stamler, J. S., Singel, D. J., Loscalzo, J., Science 1992, 258,1898–1902.

[2] Kashiba-Iwatsuki, M., Miyamoto, M., Inoue, M., Arch. Bio-chem. Biophys. 1997, 345, 237–242.

[3] Minamiyama, Y., Takemura, S., Inoue, M., Biochem. Bio-phys. Res. Commun. 1996, 225, 112–115.

[4] Gautam, A., Seligson, H., Gordon, E. R., Seligson, D., Boyer,J. L., J. Clin. Invest. 1984, 73, 873–877.

[5] Reed, R. G., J. Biol. Chem. 1986, 261, 15619–15624.[6] Gryzunov, Y. A., Arroyo, A., Vigne, J. L., Zhao, Q. et al., Arch.

Biochem. Biophys. 2003, 413, 53–66.[7] Tiruppathi, C., Naqvi, T., Wu, Y., Vogel, S. M. et al., Proc.

Natl. Acad. Sci. USA 2004, 101, 7699–7704.[8] Cha, M. K., Kim, I. H., Biochem. Biophys. Res. Commun.

1996, 222, 619–625.[9] Lee, H., Cha, M. K., Kim, I. H., Arch. Biochem. Biophys.

2000, 380, 309–318.[10] Kim, I. G., Park, S. Y., Oh, T. J., Arch. Biochem. Biophys.

2001, 388, 1–6.[11] Leonard, W. J., Jr., Vijai, K. K., Foster, J. F., J. Biol. Chem.

1963, 238, 1984–1988.[12] Bos, O. J., Labro, J. F., Fischer, M. J., Wilting, J., Janssen, L.

H., J. Biol. Chem. 1989, 264, 953–959.[13] Wanwimolruk, S., Birkett, D. J., Biochim. Biophys. Acta

1982, 709, 247–255.[14] He, X. M., Carter, D. C., Nature 1992, 358, 209–215.[15] Curry, S., Mandelkow, H., Brick, P., Franks, N., Nat. Struct.

Biol. 1998, 5, 827–835.[16] Harmsen, B. J., De Bruin, S. H., Janssen, L. H., Rodrigues

de Miranda, J. F., Van Os, G. A., Biochemistry 1971, 10,3217–3221.

[17] Rosengren, A., Bjellqvist, B., Gasparic, V., in: Radola, B. J.,Graesslin, D. (Eds.), Electrofocusing and Isotachophoresis,Wiley-VCH, Berlin 1977, pp. 165–171.

[18] Righetti, P. G., Gacon, G., Gianazza, E., Lostanlen, D.,Kaplan, J. C., Biochem. Biophys. Res. Commun. 1978, 85,1575–1581.

[19] Attanasio, F., Bruschi, M., Candiano, G., Galletto, R. et al.,Electrophoresis 1999, 20, 1403–1411.

[20] Tanford, C., Physical Chemistry of Macromolecules, Wiley,New York 1961, pp. 317–586.

[21] Ghiggeri, G. M., Cercignani, G., Ginevri, F., Bertelli, R. et al.,Kidney Int. 1991, 40, 35–42.

[22] Laemmli, U. K., Nature 1970, 227, 680–685.[23] Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst,

P., Snyder, S. H., Nat. Cell. Biol. 2001, 3, 193–197.[24] Ghiggeri, G. M., Candiano, G., Ginevri, F., Gusmano, R. et

al., Kidney Int. 1987, 32, 69–77.[25] Ghiggeri, G. M., Ginevri, F., Candiano, G., Oleggini, R. et al.,

Kidney Int. 1987, 32, 547–553.[26] Musante, L., Candiano, G., Bruschi, M., Zennaro, C. et al.,

Proteomics 2002, 2, 197–205.[27] Ghiggeri, G. M., Carraro, M., Vincenti, F., Nephrol Dial

Transplant. 2004, 19, 1036–1040.[28] Janssen, L. H., Van Wilgenburg, M. T., Wilting, J., Biochim.

Biophys. Acta 1981, 669, 244–250.


Transitions of serum albumin in patients with glomerulosclerosis ‘in vivo’ characterization by...

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