Figure 1.
Characteristics of Human serum albumin (HSA).
Human serum albumin was obtained from Human BioPlazma, Gödöllő, Hungary. The purity of the HSA was tested by SDS-PAGE first (A). There was 6 and 12 μg HSA loaded into the wells. Gel was then stained by Coomassie to visualize proteins. Apparent molecular masses of a set of standard proteins (arrows on the left) and the expected position of the HSA (arrow on the right) are indicated. Mass spectrometric (MALDI-TOF) analysis was also done (B). A representative spectrogram is shown on the figure, where the peaks representing the differently ionized HSA molecules are shown.
Figure 2.
Characteristics of recombinant ACE.
Recombinant ACE was produced with a Bac-to-Bac TOPO expression system (Invitrogen) according to the manufacturer's instructions. The pellet of baculovirus infected SF9 insect cells was homogenized in radioimmunoprecipitation assay buffer (RIPA) by sonication. The supernatant was injected onto an anion-exchange column. Column was then washed with a running buffer with different NaCl contents (shown on A). The ACE was eluted with a gradually increasing concentration of NaCl from 168 mM to 540 mM (hatched, A). Protein concentration was continuously measured (absorbance at 230 nm, green line, B) while ACE activity was measured in the collected fractions (300 μL each, red line on Panel B). Fractions with at least 50 U/L activity were combined (determined by FAPGG hydrolysis, hatched, B). Inhibitory effect of captopril was tested on the purified recombinant and on the serum ACE. Symbols represent the mean, bars are SEM of the three independent determinations.
Figure 3.
Human serum albumin interacts with the ACE.
Human serum samples were incubated with amino (-NH2) and carboxyl (-COOH) group-reactive heterobifunctional crosslinkers (succinimidyl[(n-maleimidopropionamido)-dodecaethyleneglycol] ester, SM(PEG)12, and succinimidyl[(n-maleimidopropionamido)-hexaethyleneglycol] ester, SM(PEG)6), at concentrations of 1.25, 2.5 and 5 mM (labeled +, ++ and +++, respectively) for 60 min at room temperature. Free amino groups were then blocked by TRIS (50 mM) and the adducts were immunoprecipitated and detected by an anti-ACE antibody (A). In some cases the anti-ACE antibody was omitted (first lane, A). The apparent molecular masses of ACE, the IgG heavy chain and the crosslinked product are shown on the left (A). Similar experiments were performed with purified ACE (100 kDa ultrafiltered serum) and purified HSA (24 and 48 mg/mL, labeled + and ++, respectively). Crosslinked proteins were precipitated by anti-ACE antibody (except for the first lanes, where control goat IgG was added) and detected by anti-ACE (B) and anti-HSA (C) antibodies. The positions of the IgG heavy chain, ACE and the crosslinked product are shown on the left (B), while the positions of the monomeric and multimeric HSA are shown on the right (C). Finally, the ACE-HSA interaction was also characterized by ELISA assay (D). 96-well plates were coated with HSA (1% in PBS) or with gelatine (1% in PBS) overnight. Bound proteins were incubated with SM(PEG)12 and SM(PEG)6 (each 625 μM) and recombinant ACE was then added to the wells (26 ng/well). Bars denote means ± SEM of the results of 4 independent experiments performed in triplicate.
Figure 4.
HSA inhibits serum and recombinant ACE.
Purification of ACE by filtration through 100±0.3 to 58.6±1.3 U/L (A). Effects of HSA were tested on recombinant (B) and partially purified ACE (100 kDa ultrafiltered serum; C). The ACE activity was measured by FAPGG hydrolysis. Symbols denote means ± SEM of the 3 independent experiments. Data were fitted by nonlinear regression and the calculated IC50 values are shown. The physiological HSA concentration range (35–52 mg/mL) in human serum is also indicated by green.
Figure 5.
HSA has higher affinity for the C-terminal active site of purified ACE.
Inhibition of serum ACE was tested by active site specific fluorescent substrates: Abz-SDK(Dnp)P-OH (blue bars) for the N-terminal active site, Abz-LFK(Dnp)-OH (red bars) for the C-terminal active site. Abz-FRK(Dnp)P-OH (black bars) was used as non-site specific substrate. Bars represent means ± SEM of 3 independent determinations, values are given in the percentage of control (vehicle, without ACE inhibitor).
Figure 6.
There is no dissociative small molecular weight ACE inhibitor absorbed by the HSA.
HSA was diluted to 20/mL in FAPGG reaction buffer and split into 4 fractions with identical volume. The first was used as a control (initial sample, 0 filtration cycles). The HSA was diluted in the other fractions by 10-fold and filtered through 5 kDa pore size membranes until the HSA concentration (and volume) of the retained fractions reached the original 20 mg/mL. This filtration step was repeated by 5, 10 or 15 times as indicated on the figure. The effects of these HSA fractions were tested on recombinant ACE using FAPGG substrate at a final concentration of 10 mg/mL. Maximal ACE activity was determined in the absence of HSA (vehicle). Captopril (1 μM) was also used to estimate the effect of complete ACE inhibition on FAPGG hydrolysis.
Figure 7.
HSA inhibits tissue ACE in human vascular bed.
Human saphenous vein rings were mounted on an isometric contractile force measurement setup. Vessels were treated with angiotensin peptides (angiotensin I in A) at 1 μM for an extended period of time (without washing). The vascular segments contracted, as indicated by the increase in contractile force, and then relaxed in the continuous presence of angiotensin I. At the end of the experiment, 1 μM angiotensin II was added to confirm desensitization, and the viability of the vessel was finally tested with norepinephrine (100 μM). An individual experiment is illustrated in A, force was recorded in every 0.5 s. The effects of HSA were tested on separate vascular segments. Maximal contractile responses are shown on the bar graph (B) to angiotensin peptides in the absence (Control, blue) or presence of 20 mg/mL HSA (red), in the presence of 20 mg/mL HSA plus 10 μM captopril (yellow) or norepinephrine (green). Bars denote means ± SEM, significant difference is indicated by the p value.
Figure 8.
HSA slows down the kinetics and inhibits angiotensin I evoked constrictions in saphenous vein segments.
Several parameters of transient contractile response to angiotensin peptides were investigated, including maximal force, kinetics of contraction, duration of half-maximal contraction, kinetics of desensitization and the level of desensitization (A). The responses evoked by angiotensin I (B) and angiotensin II (C) in the absence (blue) and in the presence (red) of 20 mg/mL HSA were recorded on 35 vascular segments from 20 patients. The recorded values were aligned according to the position of the maximal response and the mean ± SEM for each time point was calculated and plotted (mean, thick line, and SEM, thin line; B and C).
Figure 9.
HSA affects the rate of angiotensin evoked force development in human vascular bed.
Functional parameters such as kinetics of contraction (A), the kinetics of desensitization (B), the duration of half-maximal contraction (C), and the level of desensitization (D) were determined upon angiotensin contractions. Experiments were performed in the absence (blue) or presence (red) of 20 mg/mL HSA. Bars denote means ± SEM. Significant difference is shown by the p value.
Figure 10.
Hypothetical model for the effects of human serum albumin (HSA) on the angiotensin I converting enzyme (ACE) activity.
We found a high degree of serum ACE inhibition by physiological concentrations of HSA. This may provide a mechanism for suppressing the circulating ACE, confining angiotensin I conversion to the tissues. Vascular tissue-bound ACE was also found to be inhibited by HSA, in vitro. However, ACE inhibitor drugs are markedly effective in hypertension and heart failure. Since serum ACE is suppressed by HSA, ACE inhibitor drugs can probably not inhibit more effectively this ACE population. This can only be explained by the hypothesis that HSA does not uniformly inhibit tissue-bound ACE in the human body. Some of tissue-bound ACE can be inhibited by ACE inhibitor drugs over the inhibition provided by HSA.