N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 64

Table 5.7 Levels of N-linked Hcy in albumin and hemoglobin from different species (Data from
[297])
Specie N-Hcy/albumin, mol/mol 1,000 N-Hcy/hemoglobin, mol/mol 1,000

Human 2.65 14.1 4.1

Baboon 12.7 0.4

Bovine 3.17 1.56 30.7 0.8

Pig 3.41 1.51 16.8 5.5

Rat 4.86 1.61 82.8 29.1

Mouse 8.61 0.05

Protein N-linked Hcy occurs in serum albumin from various organisms, including
human, sheep, pig, rabbit, rat, mouse, and chicken [79]. Comparisons across the
species show that the rodent proteins contain more N-linked Hcy than the primate
proteins. For example, more N-linked Hcy is present in albumins from the rat
(0.00486 mol/mol) and the mouse (0.00861 mol/mol) than in human albumin
(0.00265 mol/mol) [297]. Rat hemoglobin contains more N-linked Hcy than human
and baboon hemoglobins (0.0828 mol/mol vs. 0.0141 mol/mol and 0.0127 mol/mol,
respectively) (Table 5.7 ). This could reflect a higher relative concentration of free
Hcy in rodents as compared to humans [304]. However, human and pig albumins
contain similar levels of N-linked Hcy, as does human and pig hemoglobins [297],
indicating that human and pigs are alike in this regard (Table 5.7 ).
About 5-15 times more N-linked Hcy is present in hemoglobin than in serum
albumin (0.013-0.083 vs. 0.0027-0.008 mol/mol, Table 5.7 ) from each species
examined, from human to rat [297]. As the in vitro rates of the modification with
Hcy-thiolactone are similar for the two proteins [78], this finding can be explained
by much slower turnover of hemoglobin compared to albumin (103 days for
hemoglobin vs. 19 days for albumin in humans). Taking the blood hemoglobin
concentration as 2.2 mM (150 mg/mL), one can calculate that the concentration of
N-Hcy-hemoglobin is 28.7-183.1

μ M, i.e., much higher than the concentration of

tHcy (4-8 M) in the normal mammalian blood. These data extend to other

mammalian species the original finding that a significant fraction of Hcy present
in human blood is circulating as N-Hcy-hemoglobin [79].
Of almost three dozen individual proteins examined, only one, human
transthyretin, has been reported not to contain N-linked Hcy [104]. However, the
detection limit of the LC-MS method used in that study was 1 % relative to total
transthyretin [104], i.e., several orders of magnitude less sensitive than that of the
HPLC with fluorescence detection method, which allows quantification of as little as
0.00006 mol N-linked Hcy/mol protein [297]. Thus, the inability to detect protein N-
linked Hcy in transthyretin is most likely due to inadequate sensitivity of the method.

μ

5.2.1.3 Collagen

Severely hyperhomocysteinemic Tg-I278T Cbs / mice (plasma tHcy ¼ 272 50

M in wild-type Tg-I278T Cbs +/+ littermates) [305] have elevated
plasma N-Hcy-protein (16.6

μ Mvs.1.9 1.6 μ

4.1 μ Mvs.1.9 1.6 μ M in wild type) [113] and