1 Free Thiol of Transthyretin in Human Plasma Most Accessible to 2 Modification/Oxidation 3 Caroline Donzeli Pereira, † Naoto Minamino, ‡ and Toshifumi Takao* ,† 4 † Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan 5 ‡ National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan 6 * S Supporting Information 7 ABSTRACT: Free-thiol(s) in proteins, especially, when located on 8 the surface of the molecule, are susceptible to oxidation/modification, 9 which may cause loss of function or an alteration in the ternary 10 structure. This suggests that the status of thiol group(s) of cysteine 11 residue(s) in a protein, i.e., free-thiol versus an oxidized/modified 12 form, in vivo, could reflect the physiological state of the molecule with 13 respect to susceptibility to oxidative stress. To address this issue, we 14 established an efficient method for isolating proteins that contain free 15 thiol groups from a complex mixture, which permits the amount of 16 free-thiol form(s) to modified/oxidized forms to be estimated. 17 Albumin, which accounts for 55% of the total plasma proteins and 18 has such a free thiol and has been reported to scavenge various 19 reactive oxygen species (ROS) in vivo. The developed method was 20 used to isolate the free form of albumin from fresh plasma. However, 21 contrary to our expectations, transthyretin (TTR), which also has a single free thiol, was found to be the major protein that was 22 the most susceptible to modification/oxidation. In addition, the free-thiol form could be separated from oxidized or modified 23 molecules, permitting the relative abundance of the free-thiol form to be estimated. The findings show that the levels of the free- 24 thiol form of TTR in plasma was significantly lowered after a hydrogen peroxide treatment, even at low concentrations (0.1 25 mM), suggesting that TTR could be a useful biomarker for monitoring a ROS imbalance in relation to various oxidative stress 26 conditions. 27 I n an organism, the global thiol-disulfide redox status is 28 defined as the total distribution of thiols and disulfides in 29 diverse cellular pools. The activity and stability of many 30 enzymes, chaperones, and transcription factors are dependent 31 on the maintenance of this status inside the various cellular 32 compartments. 1 Prokaryotes and eukaryotes have a thiol- 33 mediated reversible switching mechanism that controls many 34 cellular processes. 2 In eukaryotes, 2-3% of the total proteins 35 contain free-thiol groups and these residues are involved in 36 substrate binding or catalytic processes. 3 Moreover, these 37 residues may be subject to post-translational modification, 38 acting as mediators of redox signaling and regulation due to the 39 high reactivity of the thiol group; 4 these groups are mostly 40 observed in proteins, not in peptides sequences, 1 and are 41 amenable to various types of modifications, which raises the 42 question as to whether the free and modified forms of a protein 43 might have functions. It is, thus, important to have information 44 on the types of and degree of modification of such molecules in 45 relation to physiological or pathological events. There have 46 been many reports on the modifications of Cys residues that 47 are involved the functions of a protein. However, methods for 48 specifying the relative ratio of a free-thiol to a modified or 49 oxidized form in a sample derived from a living organism are 50 few in number. Such information is critical in terms of 51 evaluating the physiological state of a protein with respect to 52 susceptibility to oxidative stress. In order to determine the 53 fraction of a free-thiol-containing protein versus its modified 54 forms in a biological sample, we established a method that 55 permits the specific isolation of thiol-containing compounds 56 from a complex mixture and applied the methodology to 57 human plasma. About 95% of the mass of proteins in human 58 plasma are made up of only 22 proteins, 5 among which the 59 most abundant protein, albumin (3.5 g/mL), contains a single 60 free-Cys, and would be expected to be isolated as a major free 61 thiol-containing protein by the present method. In fact, 62 modifications at Cys34 of albumin have been extensively 63 studied in relation to several physiological states rationalized in 64 terms of oxidative stress. 6,7 However, the findings of our study, 65 unexpectedly, showed that transthyretin (TTR) was the major 66 trappable protein containing a free-thiol group using the 67 current method. 68 Transthyretin (TTR), formerly called prealbumin, is a 69 protein that is produced by the liver and secreted into the 70 plasma and is also found in cerebrospinal fluid. It was Received: April 27, 2015 Accepted: October 6, 2015 Article pubs.acs.org/ac © XXXX American Chemical Society A DOI: 10.1021/acs.analchem.5b03431 Anal. Chem. XXXX, XXX, XXX-XXX sac00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.6.i10:44431 | 2.0 alpha 39) 2015/07/15 14:30:00 | PROD-JCAVA | rq_5173836 | 10/09/2015 01:33:17 | 7 | JCA-DEFAULT