Η απολιποπρωτεΐνη Ε (apoE) είναι κύριο συστατικό των VLDL λιποπρωτεϊνών και των υπολειμμάτων χυλομικρών και είναι υπεύθυνη για την απομάκρυνση των αθηρογενετικών λιποπρωτεϊνών από την κυκλοφορία. In vivo και in vitro μελέτες έχουν δείξει ότι μεταλλάξεις στην apoΕ που εμποδίζουν την πρόσδεση των λιποπρωτεϊνών που περιέχουν την apoΕ στον υποδοχέα της LDL (LDLr), συνδέονται με υψηλά επίπεδα χοληστερόλης στο πλάσμα και προκαλούν πρώιμη αθηροσκλήρωση σε ανθρώπους και πειραματόζωα. Στον άνθρωπο υπάρχουν τρεις κύριες φυσικές ισομορφές της apoE που ονομάζονται E2, E3, E4 και είναι αποτέλεσμα μεταλλάξεων στα αμινοξικά κατάλοιπα 112 και 158. Προηγούμενες μελέτες σε πειραματόζωα έχουν δείξει πως η apoE διαμεσολαβεί στην εμφάνιση διατροφικά επαγόμενης παχυσαρκίας. Σκοπός της εργασίας είναι να αξιολογήσουμε το ρόλο της κάθαρσης των λιποπρωτεϊνών που περιέχουν apoE μέσω του υποδοχέα LDLr στην εμφάνιση παχυσαρκίας, καθώς οι ισομορφές Ε3, Ε4 έχουν πολύ μεγαλύτερη συγγένεια για τον LDLr από την Ε2 ισομορφή. Τα πειραματόζωα που χρησιμοποιήθηκαν ήταν πειραματικά ποντίκια αγρίου τύπου C57BL/6, ποντίκια με καθολική έλλειψη στην apoE (apoE-/- ) και ποντίκια που εκφράζουν την ανθρώπινη E2, E3, E4 ισομορφή αντίστοιχα. Τα πειραματόζωα τρέφονταν με δίαιτα δυτικού τύπου για ένα χρονικό διάστημα 24 εβδομάδων ενώ παράλληλα πραγματοποιήθηκαν βιοχημικές και μεταβολικές μελέτες. Παρατηρήσαμε πως τα ποντίκια που εκφράζουν την apoE2, παρά την χαμηλή συγγένεια που έχουν ως προς τον LDLr, αύξησαν το βάρος τους περισσότερο και εμφάνισαν υψηλότερες τιμές χοληστερόλης και τριγλυκεριδίων στο αίμα σε σχέση με τις υπόλοιπες ισομορφές. Γεγονός που αποδεικνύει πως η κάθαρση των λιπιδίων του αίματος δεν σχετίζεται με την εμφάνιση παχυσαρκίας στα πειραματικά μοντέλα ποντικών. Αντίθετα οι δοκιμασίες ανοχής στη γλυκόζη που πραγματοποιήθηκαν έδειξαν πως τα apoE3 +/+ ποντίκια εμφάνισαν τη χειρότερη ανοχή στη γλυκόζη, ενώ οι apoE2 +/+ , apoE4 +/+ ομάδες ποντικών είχαν στατιστικά παρόμοιες καμπύλες. Σύμφωνα με τα παραπάνω προκύπτει πως εμφάνιση διαβήτη και διατροφικά επαγόμενης παχυσαρκίας είναι ανεξάρτητα πεδία που πρέπει να μελετηθούν ξεχωριστά. / Apolipoprotein E (apoE) is a major component of VLDL and chylomicron remnants and is responsible for the removal of atherogenic lipoproteins from the circulation. In vivo and in vitro studies have shown that apoE mutations that prevent the binding of apoE-containing lipoproteins to LDLr, are associated with high plasma cholesterol levels and cause premature atherosclerosis in humans and animals. In humans, there are three main natural isoforms of apoE called E2, E3, E4 and is the result of mutations in amino acid residues 112 and 158. Given that previous animal studies have shown that apoE mediates the development of diet-induced obesity, the aim of this study was the evaluation of the role of apoE-containing lipoprotein's clearance by the LDLr in the development of obesity. Taking into account that the E3, E4 isoforms have higher LDLr affinity compared to the E2 isoform, we focused on the role of different apoE isoforms in these metabolic diseases. The animals we used in this study were apoE-deficient mice (apoE-/-), mice expressing human E2 (apoE2+/+), E3 (apoE3+/+), E4 (apoE4+/+) isoform and wild type C57BL/6 mice as a control group. The animals were fed western type diet for a 24-week period while biochemical and metabolic studies were performed. We observed that mice expressing apoE2, despite having low LDLr affinity, had higher body weight compared to C57BL/6 and exhibited higher plasma cholesterol and triglyceride levels compared to the other isoforms. This observation demonstrates that the clearance of blood lipids is not associated with obesity in experimental mouse models. Conversely, the glucose tolerance tests carried out showed that the apoE3+/+ mice had the worst glucose tolerance, followed by apoE4+/+ and the apoE2+/+ mice groups (apoE3+/+>>apoE4+/+≥apoE2+/+) suggesting that in case of glucose tolerance the clearance of apoE-containing lipoproteins may be a factor. Based on the above, diet-induced obesity and diabetes are, probably, independent fields that should be studied separately.

A leading cause of morbidity and mortality in the developed world is cardiovascular disease (CVD). Like many other disease processes the etiology of CVD has origins in both genetic and environmental factors. These factors affect the development of the heart and vasculature and how they respond to physiological and pathological stress. Abnormal heart development can lead to cardiac pathologies that manifest in a shift from normal cardiac geometry and physiology to what is called pathological cardiac remodeling. Often though, pathological remodeling can result from cardiovascular stress even when heart development is normal. Growth factors are essential mediators of cardiac development and physiology and a good number of clinical and experimental studies have implicated growth factors and their signaling effectors as potential therapeutic targets for pathological cardiac remodeling. Of those is Fibroblast Growth Factor 2 (FGF2) which is a potent inducer of fibroblast and cardiomyocyte proliferation in vitro. FGF2 is made in high molecular weight and low molecular weight isoforms (Hi FGF2 and Lo FGF2, respectively). It has already been demonstrated that, in the context of the heart, FGF2 modulates cardiac hypertrophy, cardiac fibrosis and mediates protection against cardiac injury. However, the isoform specific role of FGF2 in cardiac development, physiology and pathological remodeling has not been disclosed, and in this dissertation I address the hypothesis that FGF2 has isoform-specific function in cardiac physiology and remodeling. To test this hypothesis I used mice that are either deficient in Hi FGF2 (Hi KO) or Lo FGF2 (Lo KO) and subjected them to echocardiographic analysis and isoproterenol (Iso) treatment and compared them to wildtype (WT) cohorts. At baseline echocardiographic measurements, female Lo KO hearts are smaller and present with increased peak E-wave velocity, a diminutive A wave, and shortened mitral-flow deceleration time consistent with a restricted filling pattern and myocardial stiffness. Conversely, male Lo KO hearts present with a lower E wave and a higher A-wave velocity and a prolonged isovolumic-relaxation time consistent with impaired left ventricular (LV) relaxation. Female Hi KO hearts display no significant deviation from WT, while male Hi KO hearts exhibit increased systolic function. Hence, a deficiency in Lo FGF2 results in a shift from normal diastolic parameters and geometric measurements which is gender specific. Conversely, a deficiency in Hi FGF2 produces a phenotype in male hearts only. Histological and gravimetric analysis of Lo KO and Hi KO hearts post-Iso treatment reveals that female Lo KO hearts remain smaller even though their cardiomyocytes are hypertrophied while female Hi KO hearts present with a blunted hypertrophic response indicating a hypoplastic myocardium. Male Lo KO hearts present with an exacerbated fibrotic response and increased alpha-smooth muscle actin protein expression while Hi KO hearts exhibit a resistance to the fibrotic response and an induction of atrial natriuretic factor protein expression. Thus, in female hearts Hi FGF2 mediate cardiac hypertrophy while in male hearts Lo FGF2 and Hi FGF2 display an antithetical role in cardiac fibrosis where Lo FGF2 is protective while Hi FGF2 is damaging. Hence, cardiac remodeling following catecholamine overactivation is modulated by FGF2 in isoform- and gender-specific manners. In conclusion, the results presented here provide novel evidence on the interaction of gender and endogenous FGF2 isoforms as modulators of cardiac development, physiology and remodeling. Lo FGF2 signaling is necessary in the male heart for normal myocardial relaxation and for amelioration of the fibrotic response induced by beta-adrenergic stress, while in female hearts Lo FGF2 is necessary for normal cardiac growth and normal myocardial compliance. Hi FGF2 is necessary only in female hearts for mediating the hypertrophic response. Hence, I demonstrate that Lo FGF2 and Hi FGF2 have non-redundant roles in cardiac physiology and remodeling which are gender-specific.

Loss of the promyelocytic leukemia (PML) protein is associated with genomic instability/cancer. There are several isoforms of the PML protein that localize in PML nuclear bodies (PML NBs). How each individual isoform contributes to the functions of PML NBs is unknown. The objective of this study was to identify and characterize PML isoform-I (PML-I) specific protein-protein interactions. Using yeast two-hybrid screens, several interacting partners of PML-I were identified that play roles in translational regulation, including eukaryotic initiation factor 3 subunit K (eIF3K). Our studies demonstrated that eIF3K interacts with PML-I in vitro and in vivo. Through its interaction with eIF3K, overexpression of PML-I resulted in the concomitant increase in eIF3K protein levels in mammalian cells. This suggests that PML-I may be involved in regulating eIF3K protein translation or stability, which in turn could affect translation of specific mRNAs or global translation in cancer cells with reduced expression of PML-I.

Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malus x domestica Borkh.), is converted to fructose by SORBITOL DEHYDROGENASE (SDH; EC 1.1.1.14) which is active in apple fruit throughout fruit development. Apple fruit set and early development is very sensitive to carbohydrate availability, but details on carbohydrate metabolism during this phase are limited. The first objective of this work was to determine if SORBITOL DEHYDROGENASE, the primary enzyme responsible for metabolism of the major phloem-transported carbohydrate sorbitol, is present and active during apple fruit set and early development. The second objective of this work was to determine if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Nine different genes encoding SDH were determined from analysis of a cDNA library and genomic-clones. Northern, Western and ELISA analyses showed that SDH transcripts and SDH protein were present in the fruit during the first 5 weeks after bloom and comprised 7 to 8 % of the total extractable protein. Whole fruit SDH activity was highest at 2 to 3 weeks after bloom in each of three cultivars, Lodi, Redchief Delicious and Fuji. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g FW, and seed SDH activity contributed significantly to whole fruit SDH activity during the first five weeks of development after bloom. Five of the nine SDH genes present in apple genome were expressed in apple fruit (SDH1, SDH2, SDH3, SDH6, SDH9). Expression of SDH6 and SDH9 was seed-specific and expression of SDH2 was cortex-specific. Using 2D SDS-PAGE and Western analyses, SDH isomers with pI values 4.2, 4.8, 5.5 and 6.3 were found in seeds, and SDH isomers with pI values 5.5, 6.3, 7.3 and 8.3 were found in cortex. The present work is the first to show that SDH is differentially expressed and highly active in seed and cortex during early development. Thus, SDH during apple fruit set and early development may play a primary role in defining fruit sink activity.

INTRODUCTION: Persistent Pulmonary Hypertension of the Newborn (PPHN) is defined as the failure of normal circulatory relaxation in the lungs at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. Actin polymerization (APM), regulated by Rho GTPases, stabilizes force generation. We studied basal and thromboxane (TP)-induced APM and contraction in normoxic and hypoxic PA myocytes and rings. We also examined the downstream signaling pathways regulating hypoxia and TP-induced APM, and the role that actin plays in TP receptor internalization. METHODS: Smooth muscle myocytes from 2nd to 6th generation PAs of newborn piglets were cultured and exposed to hypoxia (10% O2) or normoxia (21% O2) for 72 hrs, then challenged with 10-6M TP-agonist U46619. APM was quantified by laser-scanning cytometry and stress fiber isolation. Downstream signaling pathways of TP receptor were studied by immunoprecipitation, Rhotekin-RBD and PAK-PBD affinity precipitation, Western blot, immunofluoresence and ELISA. Isometric force to serial concentrations of U46619 was measured in resistant PAs from PPHN and 3-day control swine. RESULTS: Hypoxia induced 2-fold APM via alpha- and gamma-actin isoforms, which contributed to increase U46619-induced contraction. Hypoxia decreased TP association with G12/13 in favor of Gαq. Basal RhoA and Cdc42 activity increased in hypoxia, while Rac activity decreased. U46619-challenge did not further alter RhoA activity in hypoxic cells, but increased Cdc42 and Rac activity. Hypoxia increased phosphorylation of LIMK and PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia, but increased after U46619 exposure. Jasplakinolide significantly stabilized gamma filaments, increasing force generation; cytochalasin D depolymerized all actin isoforms, which attenuated contractile force. Both actin-modifying agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. CONCLUSIONS: PA myocytes exhibit marked RhoA- and Rac-dependent APM in hypoxia. The additional APM response to U46619 challenge is independent of RhoA, but requires Cdc42 signaling. Hypoxia induces APM in PA myocytes, particularly causing an increase in filamentous alpha- and gamma-actin that contributes to increased U46619-induced force generation, a characteristic of PPHN. Dynamic actin also facilitates internalization of the TP receptor. Determining the mechanism that controls TP-mediated APM maybe beneficial as a potential target for PPHN.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph.D. in Physiology & Biophysics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 125-138). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-04-25Bitstream added on 2014-06-13T20:08:23Z : No. of bitstreams: 1 monteiro_ac_me_rcla.pdf: 419901 bytes, checksum: 6e17ba68310913d5db3d8de5269a40dd (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As pectinases são enzimas com importantes aplicações econômicas, especialmente na indústria alimentícia. Pectinases termoestáveis, produzidas por microrganismos termofílicos, apresentam uma série de características que favorecem sua aplicação em processos industriais, como, por exemplo, estabilidade em ampla faixa de pH e temperatura e maior tolerância a agentes químicos desnaturantes de proteínas. Contudo, os principais microrganismos empregados na produção de poligalacturonases (PGs) são fungos mesofílicos. O presente trabalho teve por objetivo realizar um estudo comparativo de produção de PGs em fermentação em estado sólido (FES) por fungos mesofílicos e termofílicos. Foram utilizados os fungos mesofílicos Aspergillus niger NRRL 3112 e Penicillium itallicum IZ 1584, reconhecidos como bons produtores de PGs, os termofílicos Thermomyces lanuginosus ROB e Rhizomucor pusillus A13.36 e o termodúrico Aspergillus fumigatus N31. Os microrganismos foram cultivados em FES em farelo de trigo, os mesofílicos a 27ºC e os demais a 45ºC, por períodos de 10 a 15 dias. Os maiores produtores de PG foram P. itallicum IZ 1584 (51U/g) e A. fumigatus N31 (59,40 U/g). As PGs dos fungos termofílicos apresentaram maiores valores de temperatura ótima e maior termoestabilidade que as dos mesofílicos. T. lanuginosus produziu PG com maior valor de temperatura ótima (70ºC). A. fumigatus produziu a PG mais termoestável, que conservou 100% da atividade original após 1 h de incubação a 50ºC em ausência de substrato. Cromatografias em filtração em gel e zimogramas revelaram a expressão de 7 isoformas de PG em A. niger NRRL 3112, 3 isoformas em P. itallicum IZ 1584, T. lanuginosus ROB e R. pusillus, e 2 isoformas em A. fumigatus N31. / Pectinases are enzymes with important economic applications, especially in food industry. Termostable pectinases, produced by termophilic microorganisms, display an array of characteristics that support their application in industrial processes, such as stability through a wide pH and temperature range, as well as higher tolerance to protein denaturating chemical agents. However, the major microoganisms employed in polygalacturonases (PGs) prodution are mesophilic fungi. The present work aimed to make a comparative study of PG production by mesophilic and thermophilc fungi in Solid-State Fermentation (SSF). The fungi employed were the mesophilic Aspergillus niger NRRL 3112 and Penicillium itallicum IZ 1584, both known as good PGs producers, the thermophilic Thermomyces lanuginosus ROB and Rhizomucor pusillus A13.36 and the termoduric Aspergillus fumigatus N31. The fungi were bred in wheat bran in SSF, the mesophilic at 27ºC and the others at 45ºC, during periods of 10 to 15 days. The higher PGs producers where P. itallicum IZ 1584 (51U/g) and A. fumigatus N31 (59,40 U/g). PGs of thermophilc fungi have shown higher values of optimum temperature and termostability than mesophilic PGs. T. lanuginosus produced PG with the higher value of optimum temperature for activity (70ºC). A. fumigatus have shown the most thermostable PG, which mantained 100% of its original activity after 1 h of incubation at 50ºC in absence of substrate. Gel filtration chromatography and zymograms revealed the expression of 7 PG isoforms for A. niger, 3 isoforms for P. itallicum IZ 1584, T. lanuginosus ROB and R. Pusillus, and 2 isoforms for A. fumigatus N31.

Orientador: Eleni Gomes / Banca: Eleonora Cano Carmona / Banca: Hamilton Cabral / Resumo: As pectinases são enzimas com importantes aplicações econômicas, especialmente na indústria alimentícia. Pectinases termoestáveis, produzidas por microrganismos termofílicos, apresentam uma série de características que favorecem sua aplicação em processos industriais, como, por exemplo, estabilidade em ampla faixa de pH e temperatura e maior tolerância a agentes químicos desnaturantes de proteínas. Contudo, os principais microrganismos empregados na produção de poligalacturonases (PGs) são fungos mesofílicos. O presente trabalho teve por objetivo realizar um estudo comparativo de produção de PGs em fermentação em estado sólido (FES) por fungos mesofílicos e termofílicos. Foram utilizados os fungos mesofílicos Aspergillus niger NRRL 3112 e Penicillium itallicum IZ 1584, reconhecidos como bons produtores de PGs, os termofílicos Thermomyces lanuginosus ROB e Rhizomucor pusillus A13.36 e o termodúrico Aspergillus fumigatus N31. Os microrganismos foram cultivados em FES em farelo de trigo, os mesofílicos a 27ºC e os demais a 45ºC, por períodos de 10 a 15 dias. Os maiores produtores de PG foram P. itallicum IZ 1584 (51U/g) e A. fumigatus N31 (59,40 U/g). As PGs dos fungos termofílicos apresentaram maiores valores de temperatura ótima e maior termoestabilidade que as dos mesofílicos. T. lanuginosus produziu PG com maior valor de temperatura ótima (70ºC). A. fumigatus produziu a PG mais termoestável, que conservou 100% da atividade original após 1 h de incubação a 50ºC em ausência de substrato. Cromatografias em filtração em gel e zimogramas revelaram a expressão de 7 isoformas de PG em A. niger NRRL 3112, 3 isoformas em P. itallicum IZ 1584, T. lanuginosus ROB e R. pusillus, e 2 isoformas em A. fumigatus N31. / Abstract: Pectinases are enzymes with important economic applications, especially in food industry. Termostable pectinases, produced by termophilic microorganisms, display an array of characteristics that support their application in industrial processes, such as stability through a wide pH and temperature range, as well as higher tolerance to protein denaturating chemical agents. However, the major microoganisms employed in polygalacturonases (PGs) prodution are mesophilic fungi. The present work aimed to make a comparative study of PG production by mesophilic and thermophilc fungi in Solid-State Fermentation (SSF). The fungi employed were the mesophilic Aspergillus niger NRRL 3112 and Penicillium itallicum IZ 1584, both known as good PGs producers, the thermophilic Thermomyces lanuginosus ROB and Rhizomucor pusillus A13.36 and the termoduric Aspergillus fumigatus N31. The fungi were bred in wheat bran in SSF, the mesophilic at 27ºC and the others at 45ºC, during periods of 10 to 15 days. The higher PGs producers where P. itallicum IZ 1584 (51U/g) and A. fumigatus N31 (59,40 U/g). PGs of thermophilc fungi have shown higher values of optimum temperature and termostability than mesophilic PGs. T. lanuginosus produced PG with the higher value of optimum temperature for activity (70ºC). A. fumigatus have shown the most thermostable PG, which mantained 100% of its original activity after 1 h of incubation at 50ºC in absence of substrate. Gel filtration chromatography and zymograms revealed the expression of 7 PG isoforms for A. niger, 3 isoforms for P. itallicum IZ 1584, T. lanuginosus ROB and R. Pusillus, and 2 isoforms for A. fumigatus N31. / Mestre

Aqueous Phase Partitioning has a long history of applications to the analytical characterisation of biomolecules. However process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The main aim of this work was to explore the proposition that partition in Aqueous Two Phase Systems (ATPS) can be used as an analytical tool to detect protein isoforms and to assess the applicability of the method in clinical assays and for quality control in bioprocessing through examination of several analytical problems. The work also examined the development of automated methods of system preparation and sampling techniques to determine the partition coefficient in ATPS. The study demonstrated that the geometrical form of the phase diagram co-existence curve was of crucial importance since this directly affected the accuracy with which systems of defined Tie Line Length and Mass Ratio could be constructed. The TLL %Bias (accuracy) of a theoretical system range in the PEG1000-(NH4)2SO4 system at shorter TLL (12.2) was in the range +80.6% to -100% while at a longer TLL (53.1) the %Bias (accuracy) was reduced to +0.1% to -1.9%. At the same time the MR %Bias (accuracy) at shorter TLL (12.2) was in the range +59.5% to -21.3% while at the longer TLL (53.1) this was reduced to +2.7% to -2.6%. By contrast in the PEG8000-Dextran500 system the TLL %Bias (accuracy) at shorter TLL (13.1) was in the range +3.7% to -4.12%, while at a longer TLL (31.1) the range was +0.74% to -0.67%. The MR %Bias (accuracy) at the shorter TLL (13.1) was in the range +3.6% to -3% while at the longer TLL (31.1) the range was +1.1% to -1.4%. This illustrated that it is more difficult to work with a high degree of accuracy (e.g. %Bias <5%) close to the critical point in PEG-salt systems than in PEG-dextran systems. Two different approaches were taken to examine analytical phase partitioning. In the first approach the structure of the isoforms of a model protein (ovalbumin) were altered enzymatically. Analytical methods involving Strong Anion-Exchange chromatography were developed and applied to the separation of the ovalbumin isoforms. Removal of the phosphorylated groups (dephosphorylation of ovalbumin) was undertaken using alkaline phosphatase and de-glycosylation was attempted using neuraminidase and Endo-glycosidase F. However, both enzymatic approaches to deglycosylation were unsuccessful. Dephosphorylated isoforms were successfully produced and characterised. After partitioning in ATPS a clear difference was demonstrated between the behaviour of the native and dephosphorylated forms of ovalbumin. The mean % recovery in a PEG-salt ATPS was 99.8% (± 3.59) for the naive protein and 75.6% (± 4.03) for the dephosphorylated form. On the other hand, in a PEG3350-Dextran500 system, where solubility was maintained, a significant difference in the partition coefficient (K) of native and dephosphorylated ovalbumin was found. K for native ovalbumin was 0.85 while the partition coefficient of the dephosphorylated ovalbumin was 0.61. Analysis of covariance (ANCOVA) indicated that the regression coefficients of the respective partition isotherms were significantly different (p value < 0.05). In a second approach to examine analytical phase partitioning, chemical modification of a specific target surface amino acid of another model protein (serum albumin) was used to determine the degree of conjugation of the protein and also to determine its oxidative state. The method examined the reactivity of a free surface thiol to a wide range of labels ( (a) 2-methylsulfonyl-5-phenyl -1,3,4 oxidiazole reagent, (b) N-Ethylmaleimide (NEM) reagent, (c) 5, 5’-dithiobis (2-nitrobenzoate)(DTNB) (Ellman’s reagent), (d) N-pyrenylmaleimide (NPM) reagent, (e) Fluorescein-5-maleimide (F-5-M) Reagent). Only DTNB was found to modify the surface free thiol of serum albumin in a highly specific and quantitative manner. In the course of the development of a partitioning assay for surface free thiols of serum albumin significant oxidative properties were found to be associated with poly(ethylene glycol) PEG solutions and several attempts were made to find an oxidatively safe partitioning system by including antioxidants and by removal of contaminants by freeze drying. PEG3350-Dextran500 was found to provide an oxidatively safe environment for the development of a partitioning assay for the determination of albumin free thiols. A phase partitioning assay system capable of quantitatively resolving protein associated free thiols and low molecular weight thiols from a mixture of the two was developed. Correlation coefficients (R2) for the regression of experimentally determined protein free thiols in the presence of different levels of added LMW free thiol on the known addition of protein ranged from 0.77 to 0.83. The results demonstrated that the assay could quantify and distinguish both types of thiol in a simple two-step procedure.