Biomedical subjects

J L Brash

Publications and source records attributed to J L Brash.

At least 37 records · Page 2Linked to original sources

PubMed1997

A cone-and-plate device for the investigation of platelet biomaterial interactions.

A device based on the cone-and-plate flow geometry commonly employed for viscometry was developed for the investigation of cell-surface interactions. The cone-and-plate geometry is capable of generating uniform, constant shear-rate flow fields, and control of cone rotational speed allows for easy variation of fluid shear rate. The current design is adapted for use with any material that is available in the form of a flat plate (film or coating). It also allows for replicate samples (the same or different surfaces) to be evaluated simultaneously. The device was tested under varying flow conditions for its ability to measure platelet adhesion from suspensions of washed platelets containing red cells. Collagen- and albumin-coated polymer materials were used as "standard" surfaces of known platelet reactivity (high and low, respectively). Adhesion to the collagen-coated surface was measured over a range of shear rate from 0 to 300 s(-1) and times up to 15 min. Platelet adhesion was observed to increase with increasing shear rate and time. Adhesion was significantly higher in the presence of red cells as has been observed by others. Effective platelet diffusion coefficients, calculated from the data on adhesion to the collagen surface, increased with increasing shear rate. Very little platelet adhesion to the albumin-coated surface, known to be unreactive to platelets, was observed when measured over a 15 min time period at 300 s(-1) shear rate, indicating that the device itself does not stimulate the platelets in the flow field. The data generated provide validation for this device as a simple means of measuring cell adhesion under controlled flow conditions to any smooth surface available in flat plate form.

Biocompatible Materials↗

PubMed1997

Physicochemical properties and platelet interactions of segmented polyurethanes containing sulfonate groups in the hard segment.

The adhesion of platelets to a series of segmented polyurethanes having sulfonate groups in the hard segment is reported. The polyurethanes were synthesized using sulfonated chain extenders of different structure. Analogous control materials without sulfonate groups also were studied. Adhesion was measured in vitro using washed human platelets in a carrier fluid consisting of Tyrode's buffer with apyrase, albumin, and red cells at normal concentration. The polymers were characterized by gel permeation chromatography and elemental analysis. Water absorption and thermal transitions also were determined. It was found that the sulfonated materials absorb significant amounts of water while the nonsulfonated analogs do not. The surfaces of polymer films were characterized by water contact angle and XPS. The contact angles of the sulfonated surfaces were relatively low. Platelet adhesion to the sulfonated polymers was found to be very high compared to the nonsulfonate analogs. The local environment of the sulfonate groups (different chain extenders) also appears to have an effect on platelet interactions. Albumin adsorption was high on all the materials and was not correlated with platelet adhesion. It appears from this work that platelets may have a binding site that is specific for sulfonate groups.

Adsorption↗

PubMed1997

Adsorption of immunoglobulin G and anti-factor VIII inhibitory antibody from haemophiliac plasma to derivatized polystyrenes.

Modified polystyrene resins containing sulphonate groups and tyrosyl sulphamide or tyrosyl methyl ester sulphamide groups have been investigated with respect to their potential for selective binding of anti-Factor VIII inhibitory antibodies from plasma. Adsorption of total immunoglobulin G and of a monoclonal antibody to Factor VIII was measured following addition of the radioiodinated proteins to normal plasma, plasma depleted of Factor VIII by adsorption on a resin coupled to anti-Factor VIII antibody, and haemophiliac plasma containing various levels of inhibitory anti-Factor VIII antibody. Depletion of anti-Factor VIII antibody from the haemophiliac plasmas by incubation with the resins was also measured by Bethesda assay. The modified resins and their corresponding unmodified "controls' showed similar binding of total immunoglobulin G. However, only resins containing either sulphonate or a combination of sulphonate and tyrosyl sulphamide groups showed evidence of selective adsorption of anti-Factor VIII antibody from plasma.

Adsorption↗

PubMed1997

Effect of C4-, C8- and C18-alkylation of poly(vinyl alcohol) hydrogels on the adsorption of albumin and fibrinogen from buffer and plasma: limited correlation with platelet interactions.

Polyvinyl alcohol (PVA) hydrogel was partially alkylated with short (C4), intermediate (C8) and long (C18) alkyl chains to test the hypothesis that an alkylated surface might promote enhanced interaction with albumin and thus exhibit low platelet thrombogenicity. PVA hydrogel was reacted with alkyl halides (C4, C8 or C18) and coated onto polyethylene. The effect of surface alkylation (extent of alkylation and alkyl chain length) on the adsorption of human serum albumin and fibrinogen to these surfaces was investigated in both buffer and plasma. Platelet interactions were investigated in vitro using flow cytometry methods. The maximum surface concentrations of albumin and fibrinogen adsorbed from buffer onto PVA and alkylated PVA were characteristic of monolayers. At low concentrations differences in adsorption among the surfaces appeared to be related to hydrophobicity as determined by dynamic advancing water contact angle, and to degree of alkylation as determined by angle dependent XPS analysis. Alkyl chain length dependence was not observed. Adsorption from plasma was considerably lower than from buffer, except for albumin on C8-PVA where monolayer adsorption was observed. Fibrinogen adsorption from plasma was similar on PVA, C8-PVA and C18-PVA, but was higher on C4-PVA. For albumin adsorption from plasma, the initial slope of the adsorption-concentration curve was highest for C18-PVA, suggesting higher albumin affinity despite the low degree of substitution of the C18-PVA material. These data suggest possible selectivity of the C18 alkylated PVA for albumin. Platelet studies showed that C4-PVA was the least platelet reactive (microparticle generation and P-selectin expression) of the alkyl derivatized materials.

Adsorption↗

PubMed1996

Lysis of surface-localized fibrin clots by adsorbed plasminogen in the presence of tissue plasminogen activator.

The ability of plasminogen adsorbed from buffer onto sulphonated silica glass or lysine-derivatized silica glass to lyse fibrin I clots has been investigated. Clots were formed around the test surface by adding reptilase to fibrinogen solutions in which the surfaces were immersed. Tissue plasminogen activator (t-PA) was then added and the extent of clot lysis was determined by measuring the levels of the specific plasmin cleavage product of fibrinogen, B beta 1-42 peptide. The data indicate that in the presence of t-PA, B beta 1-42 generation per mole of bound plasminogen on the lysinized material is approximately two-fold higher than on the sulphonated material. It is concluded that a preformed clot may be lysed by adsorbed plasminogen in the presence of t-PA, and that clot lysis is significantly enhanced when the plasminogen is adsorbed via its lysine binding sites.

Adsorption↗

PubMed1996

Preparation and characterization of alkylated poly(vinyl alcohol) hydrogels using alkyl halides.

A poly(vinyl alcohol) hydrogel coated onto polyethylene was partially alkylated by reaction with an alkylhalide (C4, C8, or C18) in the presence of a deprotonating agent (sodium ethoxide or potassium tert-butoxiDATE Surface coverage determined by X-ray photoelectron spectroscopy (XPS) was respectively approximately 34, 25, and approximately 8% for the C4, C8, and C18 modified surfaces. Statistically significant differences were observed in the fraction of C8 and C18 grafted alkyl groups as a function of depth (i.e. take-off angle) indicating the presence of a verticle composition gradient. All three surfaces showed maximal surface coverage of alkyl groups after 1 h reaction. At this reaction time, no further coverage was observed beyond a base/PVA ratio twenty times greater than the stoichiometric ratio. The advancing contact angle data exhibited an increase in hydrophobicity that correlated with the degree of coverage obtained by XPS: 90 +/- 1, 83 +/- 0.5, and 71 +/- 1 deg for C4, C8 and C18 alkylated PVA, and 55 +/- 2 deg for PVA respectively. Large contact angle hysteresis was observed on all three surfaces consistent with surface heterogeneity.

Alkylation↗

PubMed1994

Interactions of plasminogen and fibrinogen with model silica glass surfaces: adsorption from plasma and enzymatic activity studies.

The adsorption of fibrinogen and plasminogen from plasma to silica glass, sulfonated silica glass, and lysine-derivatized silica glass has been investigated. The data indicate that the sulfonated material has a high affinity for both fibrinogen and plasminogen, but that the ratio of plasminogen to fibrinogen is greater on the lysine-derivatized surface. The adsorption data also suggest plasminogen as a possible contributor to the fibrinogen Vroman effect, whereby initially absorbed fibrinogen is displaced from the surface. The plasmin activity of plasminogen adsorbed to the lysine-derivatized silica glass and its sulfonated precursor was assessed by both a chromogenic substrate assay and a radioimmunoassay for the plasmin cleavage product of fibrinogen, the B beta 1-42 peptide. The data indicate that 1) the adsorbed plasminogen is not inherently plasmin-like; 2) the enzymatic activity associated with the bound plasminogen is significantly enhanced on both surfaces in the presence of activator; and 3) in the presence of activator, the plasmin activity per mole of bound plasminogen on the lysinized material is approximately a factor of two greater than on the sulfonated material based on the chromogenic substrate assay, and a factor of four greater based on the B beta 1-42 radioimmunoassay. The lysinized material thus exhibits several properties that are different from its sulfonated precursor. It adsorbs more plasminogen relative to fibrinogen after the Vroman peak, and this adsorbed plasminogen appears to be in a conformation that is more readily activated to plasmin. Once activated, the surface bound plasmin shows enhanced ability to cleave either a low molecular weight chromogenic substrate or a macromolecular substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMed1993

The effect of antithrombin III-independent thrombin inhibitors and heparin on fibrin accretion onto fibrin-coated polyethylene.

Prosthetic vascular grafts become coated with a layer of fibrin that contributes to graft thrombosis and occlusion. We compared the effect of antithrombin III-independent inhibitors of thrombin with heparin for their ability to prevent fibrin accretion onto a model of a vascular graft formed in vitro by coating polyethylene tubing with thrombin bound to a layer of polymerized fibrin. Equivalent antithrombin concentrations of heparin, D-Phe-Pro-Arg CH2Cl (PPACK), recombinant hirudin (r-hirudin), and Hirulog-1 were added to barium chloride-absorbed plasma containing radiolabelled fibrinogen. Whereas, PPACK and r-hirudin persistently inhibited fibrin accretion, the inhibition by heparin was transient. Hirulog-1 had no effect on early fibrin accretion and was actually associated with enhanced accretion at 30 min (control 11.7 +/- 2.0 micrograms fibrin/cm2; Hirulog-1, 18.4 +/- 3.5 micrograms fibrin/cm2, p < 0.001). Both Hirulog-1 and r-hirudin displaced radiolabelled thrombin from the fibrin surface. Whereas hirudin-thrombin complexes are stable, Hirulog-1 produces only transient inhibition of the displaced thrombin thereby accounting for the enhanced fibrin accretion with this anticoagulant. These studies show that the antithrombin III-independent inhibitors, r-hirudin and PPACK, are more effective inhibitors of fibrin accretion onto fibrin-coated polyethylene than heparin or Hirulog-1. In addition, they emphasize the importance of determining the ability of anticoagulants to displace thrombin from fibrin and to form stable thrombin-inhibitor complexes; lack of stability of thrombin-inhibitor complexes must be countered by levels of anticoagulant that are adequate to maintain its effectiveness.

Amino Acid Chloromethyl Ketones↗

PubMed1993

Protein adsorption studies on 'standard' polymeric materials.

Adsorption of fibrinogen from buffer as a single protein and from plasma to four materials has been studied. The two NIH-NHLBI primary reference standards, filler free polydimethylsiloxane and low density polyethylene, were used along with polyvinylcholoride and cellulose materials supplied by the IUPAC Working Party. The materials were examined in both film and tubing form, except for polydimethylsiloxane which was studied only in tubing form. Adsorption was measured at room temperature using 125I-labelled fibrinogen. The order of adsorbed amounts in the single protein experiments was found to be: cellulose < PVC < PE = PDMS. Apparent adsorption affinities are in the same order. In plasma, all surfaces except cellulose showed maxima in adsorption as a function of plasma concentration after 5 min contact. This is indicative of initial adsorption followed by displacement of fibrinogen (the Vroman effect). Cellulose showed very low adsorption of fibrinogen from plasma. The Vroman maxima were more pronounced on the tubing samples than on the films, and, as for the single protein experiments, adsorption was found to be less on tubing than on film samples. A tentative interpretation of the Vroman effect data suggests that the order of procoagulant activity of the materials may be: PDMS = PE < PVC < cellulose.

Adsorption↗

PubMed1993

Identification of proteins absorbed to hemodialyser membranes from heparinized plasma.

The protein layers formed during contact of plasma with hemodialysis membranes were studied. Dialysers having membranes of cellulose acetate (CA), saponified cellulose ester (SCE), cuprophane (CUP), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) were used. Heparinized human plasma was recirculated through the dialysers for four hours. They were then rinsed and the proteins adsorbed to the membranes were eluted with 2% SDS. The yields of protein from the different membranes increased in the order: PMMA < CA < SCE < CUP < PAN. This is the probable order of increasing hydrophilicity. SDS-PAGE and Western blots were performed on the dialyser eluates. The blots were positive for most of the twenty proteins tested for. There were some interesting differences in adsorption patterns among the different membrane materials, notably for high molecular weight kininogen (HMWK), plasminogen and the C3 component of complement. HMWK was intact in the eluates from CA, CUP and SCE, whereas on PMMA and PAN there was evidence of cleavage, suggesting that activation of the contact phase of coagulation was more extensive on the latter two materials. Intact plasminogen was visible on all the blots. However, low molecular weight fragments were visible in the PAN eluates, suggesting activation of the fibrinolytic pathway. Low molecular weight fibrinogen fragments eluted from PAN membranes support this conclusion. C3 was visible in the blots obtained for all membrane materials, and the data suggest that complement is activated by all the membranes. A C3 fragment at about 30 kD (possibly C3d) was seen in the blots for the cellulosic membranes but not for PMMA or PAN.

Adsorption↗

PubMed1992

Effect of sulfonation of segmented polyurethanes on the transient adsorption of fibrinogen from plasma: possible correlation with anticoagulant behavior.

The influence of polyurethane sulfonation on fibrinogen adsorption from plasma and on plasma coagulation has been investigated. Sulfonated polyurethanes were synthesized using a two-step solution polymerization in which a diamino disulfonic acid was used as chain extender, thus incorporating sulfonate groups into the hard segments. Polymer molecular weights were determined by size exclusion chromatography and weight average values were in the range of 50,000 to 200,000. Equilibrium water uptake of solid polymer specimens was substantial and was found to increase with increasing sulfonate content. Titration of sulfonate groups allowed an estimate of the retention of free sulfonate in the polymers which ranged from 50 to 85%. Loss of free sulfonate is attributed to reaction of isocyanate with sulfonate groups during chain extension. Both surface chemistry and hydrophilicity were assessed using a combination of ESCA and water contact angle measurements. The ESCA data indicate enrichment of soft segment in the surface. Contact angles show increasing hydrophilicity with increasing sulfur content. Fibrinogen adsorption from plasma to the sulfonated polyurethane surfaces was studied using radioiodine labeling. Fibrinogen surface concentration was found to increase strongly as sulfonate content increased. Fibrinogen adsorption behavior is quite different from that of conventional unsulfonated polyurethanes in the sense that the adsorption levels are much higher and there is little displacement of initially adsorbed fibrinogen (Vroman effect). The data are interpreted in terms of two mechanisms: fibrinogen uptake (i.e., absorption) into a polymer-plasma "gel" hypothesized to exist at the surface of these materials, and adsorption in the usual sense. Thrombin times of human plasma in which polymer particles were suspended were prolonged and were found to increase with increasing sulfonate content of the polymers, suggesting that sulfonate groups confer a measure of anticoagulant activity on these materials.

Adsorption↗

PubMed1992

Polyurethanes bearing pendant amino acids: fibrinogen adsorption and coagulant properties.

Segmented polyurethanes based on 4,4'-diphenylmethane diisocyanate and polypropylene oxide and chain extended with a sulfonated diamine were derivatized by reaction of sulfonate groups in the polymer with amino acids. The chemical composition of the derivatized polymers was determined by elemental analysis. Tensile stress-strain measurements indicated a slight increase in modulus and elongation with incorporation of amino acids. Water uptake at room temperature showed little change following derivatization, but at 70 degrees C increased significantly. Water contact angles were not influenced by the presence of amino acids, but electron spectroscopy for chemical analysis data showed an increase in hard segment content in the near-surface layers so that bulk and surface compositions were more nearly the same in the amino-acid-containing materials. Fibrinogen adsorption from plasma, shown previously to be high on the sulfonated polyurethanes, was reduced by derivatization, due probably to the decrease in free sulfonate content. Thrombin times of plasma in contact with these materials were essentially the same for the derivatized and underivatized materials.

Adsorption↗

PubMed1992

Adsorption of plasminogen from plasma to lysine-derivatized polyurethane surfaces.

The adsorption of plasminogen, the principal protein of the fibrinolytic pathway in blood, to a number of solid surfaces from plasma was investigated. This study forms part of a larger project to develop a fibrinolytic surface for blood-contacting applications. Polyurethanes incorporating lysine residues were developed in an attempt to promote selective adsorption of plasminogen from plasma through lysine-binding sites in the plasminogen molecule. The adsorption of plasminogen to these surfaces as well as to glass, 'conventional' polyurethanes and precursor sulphonated polyurethanes was investigated. Adsorption from citrated human plasma diluted with isotonic Tris buffer (pH 7.4) was measured under static conditions at room temperature using radioiodinated plasminogen. The following trends were observed. (1) Adsorption increases monotonically with increasing plasma concentration and there is no suggestion of transient adsorption (Vroman effect) on any of the surfaces studied. (2) Sulphonate groups appear to have a strong effect on plasminogen adsorption as was found previously for adsorption from buffer. (3) The lysine-derivatized material having the highest lysine content may show a slight increase in plasminogen binding affinity compared to its sulphonated precursor.

Adsorption↗

PubMed1992

Delivery of passivating proteins to sutures during passage through the vessel wall reduces subsequent platelet deposition by blocking fibrinogen adsorption.

Intraluminal vascular suture material, which attracts fewer than the expected number of platelets compared with the same biomaterial exposed to blood in vitro, differs from the untreated biomaterial in that it has been passed once through the vessel wall. The mechanism by which this apparently trivial maneuver reduces platelet deposition was investigated. Polypropylene suture (7-0 Prolene) was passed through human arteries (fetal and adult), and platelet deposition to the suture was measured in a standardized perfusion chamber. Single vessel passage of the sutures reduced platelet deposition by 68 +/- 23%, which contrasts sharply with the power of prostaglandin E1 (1 microM PGE1 is sufficient to abolish platelet shape change and aggregation), which inhibited only 11% of platelet deposition to the sutures. Aspirin treatment of the vessel (to prevent PGI2 formation) or endothelial stripping (to remove the ability to produce nitric oxide) had no effect on the degree of inhibition. Passage of the suture through a vessel analogue (expanded polytetrafluoroethylene) did not inhibit platelet deposition. 125I-fibrinogen adsorption to the suture after vessel passage was reduced to a degree similar to that of platelet deposition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins eluted from vessel-passed sutures revealed bands at 66, 47, and 16 kd. Western blotting indicated the presence of large amounts of albumin and hemoglobin, a moderate amount of haptoglobin, and only trace amounts of fibrinogen. When sutures were exposed to each of these proteins in vitro before perfusion, albumin and hemoglobin were found to reproduce the effect of vessel passage alone on platelet deposition. We conclude that albumin and hemoglobin adsorb to sutures during their passage through the vessel subendothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMed1991

Interactions of proteins in human plasma with modified polystyrene resins.

Investigations are reported on the composition of protein layers adsorbed from plasma to various modified polystyrene resins. As well as polystyrene itself, polystyrene bearing sulfonate groups in the benzene rings, and polystyrene sulfonate in which the sulfonate groups were converted to amino acid sulfamide, were investigated. Some of these resins were shown in previous work to have anticoagulant properties. To study the adsorption of proteins from plasma, the resins were exposed to citrate anticoagulated human plasma for 3 h. Adsorbed proteins were then eluted sequentially by 1M Tris buffer and 4% SDS solution, and examined by SDS-PAGE. The gel patterns were similar on all resins except polystyrene. From the MWs of the gel bands, the major protein component appeared to be fibrinogen. Smaller amounts of plasminogen, transferrin, albumin, and IgG were also present. In addition, Ouchterlony immunoassay of the eluates from one resin gave positive identification of complement C3, fibronectin, IgG, and IgM. Many other minor gel bands remain unidentified. A consistent finding for all resins was the presence of plasmin-type fibrinogen degradation products though the amounts varied with resin type. It is concluded from this (and from experiments showing FDP formation when fibrinogen was absorbed to the resins, from buffer containing a trace of plasminogen) that the functional groups in these materials promote the adsorption of plasminogen and its activation to a plasmin-like molecule. It appears from the substantial quantities of fibrinogen adsorbed to these materials after 3 h exposure to plasma that the Vroman effect (giving transient adsorption of fibrinogen) is not operative on these materials. It is hypothesized that specific interactions occur between fibrinogen and sulfonate groups.

Adsorption↗

PubMed1991

The Vroman effect in tube geometry: the influence of flow on protein adsorption measurements.

The transient adsorption of fibrinogen from plasma (a manifestation of the Vroman effect), due in large part to displacement by trace proteins such as high-molecular-weight kininogen (HK), factor XII, and plasminogen, has traditionally been studied in nonflowing systems in this laboratory. This paper reports new data on adsorption in tubing geometry under laminar flow. Fibrinogen adsorption from human blood plasma and whole blood diluted to varying exents was measured on glass and polyethylene tubing. The presence of flow did not change the nature of the Vroman effect, except that the processes of adsorption and displacement, which are typically diffusion-limited in static systems, were augmented by convective transport. At the highest applied shear rates of 408 and 510 s-1, the initial adsorption rate of fibrinogen was estimated to be 5.0 X 10(-5) cm/s on both surfaces. The intrinsic rate of displacement of fibrinogen (due to the Vroman effect) at high shear rates was about ten times faster from glass than from polyethylene based on data taken 5 min after the experiment started. The rates of fibrinogen adsorption and displacement were not observed to be significantly augmented by the cellular elements of whole blood at dilutions exceeding 20:1. The consistently observed axial dependence of adsorption in static and flow experiments in tubing geometry was investigated. It was concluded that the effect results, under most conditions, from the creation of a concentration boundary layer during the displacement of the equilibrating buffer by the injected protein solution. The possibility of local depletion due to rapid adsorption during injection or the final displacement of the protein solution was concluded to make lesser contributions to axial variations in measured adsorption.

Adsorption↗

PubMed1990

Analysis of proteins adsorbed to glass from human plasma using immunoblotting methods.

Glass beads were contacted with heparinized human plasma and the adsorbed proteins eluted sequentially with 1 M tris buffer, pH 7.4, and 2% SDS. The proteins in the eluates were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting procedures using antisera to 16 common plasma proteins. All sixteen proteins were found to be present in the eluates. The immunoblots provided evidence of contact phase activation although the role of the surface could not be ascertained since some contact activation was apparent in the starting plasma. Evidence was also obtained for the occurrence of complement activation and the fibrinogen Vroman effect on the glass bead surface. Extensive degradation of high molecular weight proteins observed previously in eluates from clinically used hemodialyzers was not observed in the present in vitro plasma experiments, thus supporting the hypothesis that such degradation is due to enzymes liberated in the in vivo experiments by blood cell damage.

Adsorption↗

PubMed1989

Transient adsorption of fibrinogen on foreign surfaces: similar behavior in plasma and whole blood.

The adsorption of fibrinogen from both human whole blood and plasma to a number of "foreign" surfaces is reported. Adsorption was measured as a function of plasma or blood dilution using radioiodine labeling. We showed previously that adsorption of fibrinogen from plasma exhibits a maximum at a plasma dilution of about 100:1, and have attributed this behavior to competition from other plasma proteins. (The same phenomenon is manifest as a time transient in fibrinogen adsorption.) In the present work we show that exactly the same trends are observed in whole blood. For each of the four surfaces, glass, siliconized glass, collagen-coated glass and polyethylene, the adsorption of fibrinogen as a function of dilution is the same in whole blood as in plasma. Each of these surfaces shows a unique dependence of fibrinogen adsorption on plasma or blood dilution. On cuprophane and a hydrophilic polyether urethane there is essentially no adsorption of fibrinogen from blood or plasma. For the hydrophilic polyurethane this result may be artifactual, but the absence of fibrinogen binding to cuprophane in blood or plasma is real since fibrinogen is found to be adsorbed in monolayer amounts from buffer.

Adsorption↗