Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis.
Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors --glycation of HSA--occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSA(GLC)) with HSA glycated by fructose (gHSA(FRC)). We focused on presenting the differences between gHSA(FRC) and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335nm and λem 420nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSA(FRC) is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSA(FRC) becomes less accessible for the negatively charged quencher (I(-)), KSV value is smaller for gHSA(FRC) than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum albumin, while KSV values for gHSA-KP systems are only slightly lower than that obtained for HSA-KP. The affinity of PHB to the glycated HSA is stronger than to the non-glycated in the first class binding sites within subdomain IIA, in the vicinity of Trp-214. Ketoprofen bound to unmodified human serum albumin stronger than for glycated albumin and one class of binding sites is observed (Scatchard linear plots).
Medical University of Silesia, Faculty of Pharmacy, Department of Physical Pharmacy, Katowice, Poniatowskiego 15, Poland.
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Szkudlarek
Medical University of Silesia, Faculty of Pharmacy, Department of Physical Pharmacy, Katowice, Poniatowskiego 15, Poland. Electronic address: sulkowskaanna@yahoo.com.
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Sułkowska
Medical University of Silesia, Faculty of Pharmacy, Department of Physical Pharmacy, Katowice, Poniatowskiego 15, Poland.
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Maciążek-Jurczyk
Medical University of Silesia, Faculty of Pharmacy, Department of Physical Pharmacy, Katowice, Poniatowskiego 15, Poland.
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Chudzik
Medical University of Silesia, Faculty of Pharmacy, Department of Physical Pharmacy, Katowice, Poniatowskiego 15, Poland.
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Równicka-Zubik
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D017127:Glycation End Products, Advanced / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007660:Ketoprofen / Q000378:metabolism; D008958:Models, Molecular; D009282:Naphthalenesulfonates / Q000737:chemistry; D010653:Phenylbutazone / Q000378:metabolism; D000091402:Prohibitins; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D000093423:Glycated Serum Albumin
D000894:Anti-Inflammatory Agents, Non-Steroidal; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D009282:Naphthalenesulfonates; C000717328:PHB protein, human; D000091402:Prohibitins; D012709:Serum Albumin; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D007660:Ketoprofen; D010653:Phenylbutazone; D000093423:Glycated Serum Albumin
2-(p-Toluidino)naphthalene-6-sulfonic acid; Acrylamide; Glycated human serum albumin; Ketoprofen; Phenylbutazone; Potassium iodide