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Clinical Chemistry 52: 2258-2264, 2006. First published October 26, 2006; 10.1373/clinchem.2006.074302
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2006;52:2258-2264.)
© 2006 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Bound Homocysteine, Cysteine, and Cysteinylglycine Distribution between Albumin and Globulins

Glen L. Hortin1,a, Nitin Seam2 and Gerard T. Hoehn2

Departments of1 Laboratory Medicine and 2 Critical Care Medicine, Warren Magnuson Clinical Center, National Institutes of Health, Bethesda, MD.

aAddress correspondence to this author at: Laboratory Medicine, Warren Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892. Fax 301-402-1885; e-mail ghortin{at}mail.cc.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Major portions of homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly), and glutathione in serum are covalently bound to proteins via disulfides. Albumin has been considered the dominant binding protein.

Methods: Pooled serum and plasma from healthy adults were fractionated into albumin and globulins by affinity columns. Content of Hcy, Cys, CysGly, and glutathione was determined for serum and plasma fractions and purified proteins by an HPLC method before and after incubation with excess CysGly, Hcy, or glutathione

Results: Of protein-bound amino acids in pooled serum, 12% of Hcy, 21% of Cys, and 33% of CysGly were bound to globulins, with the remainder bound to albumin. Slightly higher proportions were bound to globulins in pooled plasma. Globulins had ~16% of total exchangeable disulfide and thiol groups in serum based on results of loading with CysGly. These results agree with expected abundance of unpaired Cys residues in globulins relative to albumin. Significant amounts of disulfide-linked amino acids were detected for HDL and {alpha}1-acid glycoprotein but not for transferrin. Exchange of disulfide-linked amino acids on exposure to excess Hcy or glutathione was much faster for albumin than for {alpha}1-acid glycoprotein.

Conclusions: Approximately 10%–30%, of protein-bound Hcy, Cys, and CysGly are disulfide-linked to globulins. Amino acids disulfide-linked to albumin are rapidly exchangeable, while exchange of disulfide-linked amino acids from globulins, such as {alpha}1-acid glycoprotein, is much slower. Consequently, the pools of Hcy, Cys, and CysGly bound to albumin and globulin may represent kinetically and functionally distinct pools. Plasma concentrations of total Hcy and Cys, which are dominated by albumin-bound pools, may not reflect the abundance of functionally significant modifications of globulins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Plasma concentrations of homocysteine (Hcy) 1 and, to a slightly lesser extent, cysteine (Cys) have been correlated with risk for a variety of cardiovascular and neurologic diseases (1)(2)(3)(4)(5). These findings have stimulated clinical interest in the measurement of Hcy and Cys and the molecular forms and interactions of Hcy and Cys (5)(6)(7)(8)(9). In plasma these amino acids occur predominantly in disulfide-linked forms, most linked to protein, a smaller fraction linked to Cys, Hcy, cysteinylglycine (CysGly), or glutathione, and a very small fraction as a thiolactone (5)(6)(7)(8)(9). Small amounts of Hcy become irreversibly bound (amide-linked) to proteins via reaction of Hcy thiolactone, but most Hcy and other thiol-containing amino acids or peptides are linked to protein by reversible disulfide linkages. Only a small proportion of thiol-containing amino acids occur in reduced form, and these forms rapidly oxidize to form disulfides in blood removed for analysis (5)(6). An unpaired Cys, residue 34 in albumin, has been considered the predominant site in proteins forming disulfide linkages with thiol-containing amino acids (5)(6)(7)(10). Recent studies have determined that some Hcy and Cys are linked to lipoproteins (11) or to unpaired Cys residues in transthyretin (12)(13)(14)(15)(16)(17)(18). These findings suggest the binding of Hcy and Cys to a variety of proteins via disulfide linkages (11)(12)(13)(14)(15)(16)(17)(18). The present study sought to determine the proportions of thiol-containing amino acids and peptides bound to albumin and globulins. Recent development of immunoaffinity columns for albumin depletion of serum offers a method to efficiently fractionate serum into albumin and globulin fractions for subsequent analysis (19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chicken IgY-antialbumin microbeads obtained from GenWay Biosciences were packed into spun columns with a packed volume of 1 mL. We obtained the purified plasma components transferrin, IgG, {alpha}1-acid glycoprotein, and {alpha}1-antitrypsin from Sigma Chemical and human plasma IgA from Calbiochem. HDLs, purified by CsCl gradient centrifugation followed by extensive dialysis (20), were provided by Dr. Alan Remaley in the Department of Laboratory Medicine (Warren Magnuson Clinical Center, National Institutes of Health, Bethesda, MD). Blood collected from 19 healthy adults into EDTA-containing tubes was processed immediately, pooled, and stored frozen at –70 °C. A serum pool was formed from blood collected from 4 healthy adults into plain red-top tubes and allowed to clot for 30 min at room temperature before centrifugation to collect serum. Specimens were collected from volunteers in accordance with a protocol approved by an institutional review board. Partitioning of plasma and serum into albumin and globulin fractions was performed with IgY-antialbumin affinity spun columns. Aliquots of 20 µL of serum were diluted with 480 µL saline, pH 7.4, and mixed with the solid phase in the antialbumin column. Unbound globulins were eluted from the spun columns, and columns were washed with saline before elution of bound albumin with 100 mmol/L glycine, pH 2.5, followed by neutralization of the eluate with one-tenth volume of 1 mol/L Tris, pH 8.0. Two successive elutions with the pH 2.5 buffer were pooled. The globulin and albumin fractions of serum and plasma were prepared by pooling unbound and bound fractions from 16 runs of the serum pool and 12 runs of the plasma pool. Pooled globulin and albumin fractions were concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Millipore) with a 30 000-Da cutoff. Low molecular mass components were removed by serial dilution of specimens (~20-fold) 3 times with saline followed by ultrafiltration. Final volume was adjusted to 500 µL. Total protein concentrations were determined by a biuret method and albumin by an immunoturbidimetric method on an LX-20 analyzer (Beckman Coulter).

Thiol-containing amino acids were analyzed with cystamine as an internal standard as previously described (21). Specimens were reduced with tris(2-carboxyethyl)phosphine. Proteins were precipitated with tricholoracetic acid, and amino acids in the supernatant were derivatized with 7-flouoro-1,3-benzoxadiazole-4-sulfonate. Reaction products were analyzed by HPLC with fluorescence detection. Loading of proteins with an excess of CysGly to provide an estimate of capacity for forming disulfides with thiol-containing amino acids was performed in 125 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, 0.6 mmol/L CysGly, 0.02% sodium azide, with proteins at a concentration of 2 g/L. After incubation at room temperature for 1 day, unbound amino acids were removed by multiple exchanges with saline in Centricon-30 centrifugal concentrators (Millipore). Experiments examining the kinetics of exchange of thiol-containing amino acids were performed at room temperature in 120 mmol/L NaCl, 20 mmol/L sodium phosphate, pH 7.4, and 0.5 mmol/L EDTA. At selected time-points, aliquots of exchange reactions were diluted 10-fold with 200 mmol/L ammonium acetate, pH 5, to stop exchange reactions, and free amino acids were removed by ultrafiltration. Bound thiol-containing amino acids were then analyzed by HPLC.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The use of albumin depletion columns allowed relatively efficient separation of serum proteins into albumin and globulin fractions in a single step (Table 1 ). Albumin made up >96% of the total protein content of the albumin fraction and <4% of the total protein in the globulin fraction. Analysis of the Cys, Hcy, CysGly content of the globulin fraction showed significant amounts of each of these components. Chromatograms of the fluorescent derivatives of these thiol-containing amino acids/peptides are shown in Fig. 1 for equivalent amounts of total protein for the globulin and albumin fractions. In some analyses, there was incomplete separation of the peaks for CysGly and glutathione, and values were expressed only for CysGly because glutathione contributed only ~10%–15% to peak areas for CysGly. The contents of amino acids disulfide linked to albumin or globulins in the serum pool were calculated based on the serum concentrations of globulin and albumin (Table 1 ). Values for the globulin fraction were corrected for 3.6% content of albumin. These calculations suggest that 21% of protein-bound Cys, 12% of Hcy, and 33% of CysGly were bound to globulins. Glutathione was also released from the globulin fraction, but the peak was not well resolved from the CysGly peak, so it was not quantified in this experiment.


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  		Table 1. Content of thiol-containing amino acids and peptides [(mean (SD) of triplicates] in albumin and globulin fractions of pooled serum with a total protein concentration of 73 g/L and albumin concentration of 49.6 g/L.


Figure 1
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  		Figure 1. Chromatograms of fluorescent derivatives of thiol-containing amino acids released from globulin (top panel) or albumin (bottom panel) fractions of serum.

Peaks are indicated as Cys (cysteine), Hcy (homocysteine), CysGly (cysteinylglycine), and GSH (glutathione). The vertical axis shows fluorescence detector response in millivolts.

Total capacities of the albumin and globulin fractions to form disulfide linkages with amino acids were estimated by loading the proteins with excess CysGly and removing unbound CysGly by ultrafiltration. The total content of disulfide-linked amino acids increased by ~2-fold for both fractions, and calculations based on serum concentrations indicated that globulins represented ~16% of the total binding capacity for disulfide-linked amino acids.

The fractionation experiment was repeated for pooled plasma (Table 2 ). Albumin and globulins were efficiently separated from the pooled plasma. The globulin fraction contained <0.5% albumin, and the albumin fraction was ~97% albumin as assessed by the ratio of albumin to total protein. The globulin fraction of plasma was found to contain significant amounts of Cys, Hcy, CysGly, and glutathione. Bound components on globulins were estimated to represent ~15% of bound Hcy, 34% of bound Cys, 46% of bound CysGly, and 50% of bound glutathione in plasma. Analysis of ultrafiltrate from the final exchange step used to remove any unbound amino acids served as a blank and showed almost undetectable concentrations of the thiol-containing amino acids. This showed that the thiol-containing amino acids recovered in the globulin fraction were not due to contamination of buffers or to carryover from one analysis to the next. Good reproducibility on multiple analyses provided evidence that carryover between specimens did not account for recovery of thiol-containing amino acids in analyses of the globulin fraction.


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  		Table 2. Protein-bound thiol-containing amino acids and peptides [mean (SD) of triplicates] determined for albumin and globulin fractions of pooled plasma. Free amino acids were removed by ultrafiltration.

Several purified proteins were tested for content of disulfide-linked amino acids (Table 3 ). Before analysis, any free amino acids in the protein preparations were removed by ultrafiltration. Transferrin was considered to be a negative control, because its sequence does not contain an unpaired Cys residue. IgG also is not considered to have an unpaired Cys residue, although some unpaired Cys residues might be expressed in variable sequence elements. Disulfide-linked amino acid concentrations substantially above background values for proteins serving as negative controls were obtained for HDL and {alpha}1-acid glycoprotein. In additional experiments, 10 g/L IgA was found to contain mean (SD) concentrations of 28 (0.4) µmol/L Cys and 1.04 (0.05) µmol/L Hcy. Negligible amounts of Cys (<1 µmol/L) and Hcy (<0.3 µmol/L) were detected bound to 10 g/L {alpha}1-antitrypsin, which contains a single Cys residue. This residue, however, may be chemically modified or inaccessible, or bound amino acids may be lost during the process of purifying this protein. The values measured in these experiments should be considered minimal values for amounts of disulfide-linked amino acids bound to these proteins in the circulation, because the purification procedures for these proteins involve multiple steps during which some of the disulfide-linked amino acids might be lost.


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  		Table 3. Content of thiol-containing amino acids and peptides released from specific protein preparations by reduction.

After incubation of purified proteins with excess CysGly, most of the Cys and Hcy that were disulfide-linked to specific protein components were displaced (Table 3Up , bottom). There was an increase in CysGly linked to {alpha}1-acid glycoprotein, to HDL, and to a lesser extent, to IgG. After loading with CysGly, the total content of disulfide-linked amino acids was similar to preloading values, primarily representing an exchange of CysGly for Cys.

The rates of exchange of disulfide-linked amino acids bound to albumin and {alpha}1-acid glycoprotein were examined in the presence of excess Hcy (Fig. 2 ) or glutathione (see Table 1Up in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue11). Excess Hcy displaced Cys and CysGly linked to albumin relatively quickly, with a 50% decrease in ~30-min. Concurrently, there was an increase in albumin-linked Hcy, with half-maximal increase similarly occurring in ~30 min. Under the same conditions, there was a much slower decline in Cys and CysGly linked to {alpha}1-acid glycoprotein; ~1 day was required for a 50% decrease of disulfide-linked Cys and CysGly. Half-maximal increase in Hcy disulfide-linked to {alpha}1-acid glycoprotein required several hours, although the reaction may not have reached a final maximal endpoint at the last time-point (24 h) tested. Exchange reactions in which excess glutathione was used to exchange other disulfide-linked amino acids from albumin and {alpha}1-acid glycoprotein showed similarly more rapid displacement of Cys, Hcy, and CysGly from albumin than from {alpha}1-acid glycoprotein (see Table 1Up in the online Data Supplement). Therefore, the results indicate that, at a physiological pH, exchange of amino acids disulfide-linked to albumin is generally much more rapid than exchange of amino acids disulfide-linked to {alpha}1-acid glycoprotein,


Figure 2
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  		Figure 2. Exchange of protein-bound Cys, CysGly, and Hcy in the presence of 0.5 mmol/L Hcy.

Reactions of components bound to albumin are shown as dashed lines and components bound to {alpha}1-acid glycoprotein are shown as solid lines. For the top and middle panels, values are plotted as the percentage vs initial values, for Cys and CysGly, respectively. In the bottom panel, absolute concentrations of Hcy are plotted.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cys, Hcy, CysGly, and glutathione occur in plasma predominantly as disulfide-linked forms bound to proteins (5)(6), and albumin has been considered to account for virtually all of the protein binding (5)(6)(7)(10). However, there are several major plasma proteins besides albumin that contain unpaired Cys residues that may be available for disulfide formation with Cys and Hcy. Mass spectrometry has provided clear evidence that some of these proteins, such as transthyretin, apolipoprotein A-II, and apolipoprotein E, circulate as forms with amino acids or peptides attached via disulfides to the proteins (12)(13)(14)(15)(16)(17)(18)(22). Major plasma proteins with unpaired Cys residues and reference concentration intervals in healthy persons are listed in Table 4 . The positions of the unpaired Cys residues and the total number of Cys residues in the proteins also are noted. Not all of the unpaired Cys residues in these proteins may be available for disulfide formation with thiol-containing amino acid, because some of these unpaired Cys residues, such as in apolipoprotein A-II, are observed to form disulfide-linked homodimers or heterodimers with other proteins (18)(20)(22)(23)(24). A portion of IgA and prothrombin molecules form disulfide bonds with {alpha}1-microglobulin (25). {alpha}1-Acid glycoprotein may be one of the more abundant plasma proteins, besides albumin, with an unpaired Cys, but there are 2 genes encoding this protein, and only the gene 2 contains an unpaired Cys (26). The gene 2 product appears to represent approximately one third of the total mass of this protein (27). The gene 2 product is assumed to represent approximately one third of the total in plasma (Table 3Up ). {alpha}1-Antitrypsin is another relatively abundant protein with an unpaired Cys, although our experiments were unable to confirm the presence of Cys and Hcy linked to it via disulfides.


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  		Table 4. Reported serum concentrations in healthy persons of major serum proteins with unpaired Cys residues (18)(35)(36)(37).

The sum of several major proteins with unpaired Cys residues suggests that globulins have a potential capacity of ~100 µmol/L for disulfide-linked amino acids vs ~650 µmol/L for albumin. The relative concentrations of unpaired Cys in globulins and albumin therefore suggest that ~15% of disulfide-linked amino acids will be bound to globulins, consistent with our measures of endogenously bound disulfide-linked amino acids and peptides in serum and of total capacity for disulfide-linked amino acids assessed by loading with excess CysGly.

The unpaired Cys, residue 34, in albumin is unusual in having a thiol group with a pK <6 (7)(10). Consequently, at neutral pH, the thiol group will be predominantly in the thiolate form, which is the reactive form in the formation of disulfides (7), a situation that contrasts with the thiol groups of most Cys residues, which have pKs >8 and are predominantly in an uncharged form at neutral pH. This difference suggests that the unpaired Cys in albumin may be a more rapidly reactive and exchangeable site than unpaired Cys residues in other plasma proteins. Cys-34 of albumin also is in a relatively sterically constrained pocket (28). Steric constraints might favor reactivity of smaller reactants such as Cys and Hcy vs CysGly, glutathione, or unpaired Cys in other proteins. We observed higher ratios of Hcy:Cys and Hcy:CysGly for albumin than globulins. It will be of interest for future studies to confirm whether this is a consistent observation. Different Hcy:Cys and Hcy:CysGly ratios in albumin and globulins may reflect differences in the reactivity or accessibility of the unpaired Cys in albumin and globulins. Previous studies using mass spectrometry to analyze transthyretin have revealed surprisingly high proportions of disulfide-bound CysGly and glutathione relative to Cys, considering the total plasma concentrations of these components. One possible explanation is that the amino acids/peptides disulfide-linked to globulins may be a slowly exchangeable pool (related to high pK of their unpaired Cys residues), and that some of the glutathione may be bound intracellularly before protein secretion (29). A second possibility is that a pathway for enzymatic transfer of CysGly to proteins by gamma-glutamyltransferase (30) may be directed selectively to globulins due to the relative inaccessibility of the unpaired Cys residue in albumin. The latter possibility might account for higher CysGly:Hcy in globulin, but probably would not explain higher glutathione:Hcy.

There are several examples described in which either the disulfide linkage of unpaired Cys residues in a protein with amino acids or with other proteins influences the biological activity of a protein. The tendency of amyloidogenic molecular variants of transthyretin to deposit as amyloid is influenced by formation of disulfides with amino acids (16). Fibronectin and coagulation factor V have been suggested as targets of Hcy binding that may result in altered function (8). High circulating concentrations of Hcy appear to influence the permeability and resistance to lysis of fibrin clots (31)(32), possibly through disulfide-linkage to various plasma proteins. The molecular variant apolipoprotein A-I Milano, which introduces a single Cys residue into the protein, has a strong protective effect against atherosclerosis (23)(24). This effect has been ascribed to an antioxidant effect of the added thiol group, but it is also observed that apolipoprotein A-I Milano forms homodimers and heterodimers with apolipoprotein A-II, which may contribute to changes in function (23)(24). A variant of fibrinogen with an additional Cys in the {gamma}-chain of fibrinogen, fibrinogen Milano VII, forms heterodimers with albumin and thus may contribute to delayed clot formation (33). Apolipoprotein E is a polymorphic protein with common variants with 0, 1, or 2 Cys residues (34). These substitutions appear to have significant functional effects and disease associations, although it is not clear whether these are related to variable capacity of the different forms of apolipoprotein E to form disulfides with other proteins or amino acids.

The above examples suggest 2 potential mechanisms by which disulfide bond formation of amino acids with unpaired Cys residues may considerably alter the function of proteins. A possible direct mechanism is modification of Cys residues leading to alteration of the structure of proteins, and an indirect effect may occur through competition with other proteins for reaction with unpaired Cys residues, altering the balance of cross-linking between proteins. Examining whether changes in Hcy or Cys concentrations are correlated with altered dimerization of proteins such as apolipoproteins A-II, apolipoprotein D, or {alpha}1-microglobulin in plasma may offer a method to test whether this potential mechanism accounts for some of the pathophysiology associated with changes in Hcy and Cys concentrations.

The present study shows that a subfraction of protein-bound Cys, Hcy, CysGly, and glutathione resides on globulins. Also, the globulin-bound subfraction of these disulfide-linked amino acids and peptides may be a kinetically distinct pool with much slower turnover and exchange than amino acids disulfide-linked to albumin. The existence of 2 or more distinct pools of protein-bound Hcy and Cys offers the possibility that different pools may have different pathophysiological significance, analogous to different implications for the amounts of cholesterol bound to HDL and HDL fractions. Further study will be required to assess whether fractionation of protein-bound Hcy and Cys linked to specific proteins or analysis of slowly and rapidly exchangeable pools offers additional information about risk or pathogenesis of cardiovascular disease.


   Acknowledgments
 
Maureen Sampson assisted with the figures. Ray Kenney, Bonnie Meilinger, Patricia Sullivan, Debra Burton, and Lashea Davis provided technical assistance. Dr. Steven Drake prepared the plasma pool. Studies were supported by the intramural research program of the NIH Clinical Center.


   Footnotes
 
1 Nonstandard abbreviations: Hcy, homocysteine; Cys, cysteine; CysGly, cysteinylglycine.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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