Gillian Sudlow

Bio: Gillian Sudlow is an academic researcher from St. Vincent's Health System. The author has contributed to research in topics: Human serum albumin & Plasma protein binding. The author has an hindex of 9, co-authored 9 publications receiving 3083 citations.

The characterization of two specific drug binding sites on human serum albumin.

Abstract: The binding of a number of fluorescent probe molecules to human serum albumin (HSA) has been studied. Small changes in the amino acid moiety of the dansylamino acids resulted in large changes in the binding of these compounds to HSA. It is suggested that electrostatic and dipolar forces play a role in the specificity and binding affinity of such compounds. Fluorescent probes which had one tight binding site were used for drug displacement studies. Changes in probe fluorescence were shown, by equilibrium dialysis and by fluorescence titrations, to be a result of competitive displacement by drugs. The pattern of displacement of probes by drugs enabled the identification of two specific drug binding sites. The relative affinity of drugs for these binding sites was measured by their ability to displace fluorescent probes specific for the sites. The method provides a rapid and simple means for detecting potential drug interactions based on competition for protein binding sites.

Further characterization of specific drug binding sites on human serum albumin.

Abstract: The fluorescent probes 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine function as specific markers for two distinct binding sites for anionic drugs on human serum albumin (HSA). The binding of drugs to site I or II was detected by measuring the displacement of these probes. The results give an indication of some of the structural features required for binding at these sites. Stearic acid binding to HSA induced different conformational changes in the albumin at sites I and II, which were detected by changes in the fluorescence and/or strength of binding of probes specific for the two sites. This provides further evidence for qualitative differences between the two sites. The specificity of the sites for particular drugs, even at high drug to albumin ratios, was established, and the specificity and characteristics of the two sites on human adult and neonatal sera were shown to be similar to those on crystalline HSA.

Effects of Fatty Acids on Two Specific Drug Binding Sites on Human Serum Albumin

Abstract: Two distinct binding sites, I and II, for anionic drugs on human serum albumin have previously been demonstrated using fluorescent probe techniques. 5-Dimethylaminonaphthalene-1-sulfonamide (DNSA) and dansylsarcosine are specific fluorescent probes for sites I and II, respectively. The addition of fatty acids results in differing effects at the two binding sites, and the specificity of site II is lost. The order of potency of various fatty acids in causing these changes is oleic > stearic > linoleic [unknown] palmitic, and this is the same as the order of association constants for these fatty acids. It is concluded that chain length and degree of unsaturation determine both binding affinity and the extent to which the fatty acids induce configurational adaptations in the albumin molecule. Furthermore, studies with varying ratios of oleic acid to albumin suggest that the conformational changes induced in the protein are different for each molecule of oleic acid added. Addition of oleic acid at a 3:1 molar ratio with albumin significantly increased the binding of warfarin and DNSA to site I.

Spectroscopic techniques in the study of protein binding. A fluorescence technique for the evaluation of the albumin binding and displacement of warfarin and warfarin-alcohol.

Abstract: 1. The binding of racemic mixtures of warfarin and warfarin-alcohol to human serum albumin (HSA) is accompanied by an increase in the fluorescence quantum yield of these compounds. This property has been used to measure the characteristics of the binding of warfarin and warfarin-alcohol to HSA at 22 degrees C and 37 degrees C. Within the limits of the technique, no significant differences between the number of binding sites and strength of binding at the tight site at either temperature were observed. 2. The fluorescence of warfarin and warfarin-alcohol was used to label their binding site on HSA and to study the effects of other drugs on their binding. The results indicate that these two molecules are bound to the same site on HSA. 3. The validity of using changes in the fluorescence of warfarin as a measure of its displacement from HSA was investigated. Good correlations were observed between drug-induced decreases in the fluorescence of bound warfarin and displacement as measured by equilibrium dialysis. The displacement of warfarfin, as detected by fluorescence, correlates well with the increase in free warfarin resulting from addition of therapeutic drug concentrations to undiluted human serum. 4. The most potent displacing agents, by all the methods used, were iophenoxic acid, phenylbutazone and oxyphenylbutazone. The first of these is no longer used clinically, but the latter two are and have been reported to cause hypoprothrominaemia by displacing warfarin from HSA. The present study indicates that changes in the fluorescence of warfarin bound to HSA can be used to measure displacement of bound warfarin and to screen drugs that may cause clinically significant interactions by this mechanism.

Structure of serum albumin.

Abstract: Publisher Summary This chapter provides an insight of the findings of past significant papers with the current knowledge of the recently determined high resolution X-ray structure of serum albumin. The most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands. The sequences of all albumins are characterized by a unique arrangement of disulfide double loops that repeat as a series of triplets. Albumin belongs to a multigene family of proteins that includes α- fetoprotein (AFP) and vitamin D-binding protein (VDP), also known as G complement (Gc) protein. Although AFP is considered the fetal counterpart of albumin, its binding properties are distinct and it is suggested that AFP may have a higher affinity for some unknown ligands important for fetal development. Domain structure and the arrangement of the disulfides, the surface charge distribution, and the conformational flexibility of the albumin molecule are described. The nature of ligand binding, including small organics, long-chain fatty acids, and metals, to multiple sites on the albumin molecule is clearly depicted. The chapter concludes with the perceptive comments on future directions being taken to explore the structure and function of this fascinating protein.

Crystal structure of human serum albumin at 2.5 A resolution.

Abstract: A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.