Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells

The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF.     

Analytical and preparative electrophoresis of RNA in agarose-urea.

Agarose-6 m urea gels at neutral pH stained with ethidium bromide give high resolution of complex mixtures of RNA molecules. These RNA species can be readily distinguished from contaminating DNA, which does not have to be purified away from the RNA, and electrophoresis can be carried out using phenol-saturated deproteinated cellular extracts without loss of resolution. Individual RNA species can be extracted from the gels by freezing, thawing, and centrifugation; the RNA may then be purified by phenol extraction and ethanol precipitation. Such purified RNA is an excellent substrate for RNA-DNA hybridization and cell-free translation. In addition, the RNA can be easily transferred with high resolution from the agarose-6 m urea gels to diazobenzyloxymethyl paper for subsequent hybridization to labeled DNA.     

Glycoproteins from the cuticle of the Atlantic shore crab Carcinus maenas: I. Electrophoresis and Western-blot analysis by use of lectins

The protein and glycoprotein content of four different neutral or acidic solvent extracts (0.5 M KCl, 10% EDTA, 0.1 N HCl, or 2% acetic acid) from the mineralized exoskeleton of a decapod crustacean, the Atlantic shore crab Carcinus maenas, were characterized by quantitative analysis of proteins, SDS-PAGE analysis, and probing with lectins on blots. The lectins used were Conconavalin A, Jacalin, soybean agglutinin, Maackia amurensis agglutinin II, and Sambucus nigra agglutinin. The results show that many proteins can be obtained from the crab cuticle without strong denaturants in the extraction medium. Many of the extracted cuticle proteins appeared to be glycosylated, bearing O-linked oligosaccharides and N-linked mannose-rich glycans. N-acetyl-galactosamine and N-acetylneuraminic acids were revealed, for the first time, as terminal residues on N-linked mannose-rich structures of crab cuticle glycoproteins. Sialylated glycoproteins might thus be involved in organic-mineral interactions in the calcified crab exoskeleton. The amount and variety of glycoproteins extracted with the acidic solvents are obviously different from those extracted with neutral solvents. HCl proved to be the best of the tested extraction solvents and a valuable alternative to EDTA.     

Effects of acidity on acute toxicity of aluminium-waste and aluminium-contaminated soil

The total heavy metal concentrations of Al-waste and Al-contaminated soil were many times higher than that found in the control soil, which might pose toxic effects on nearby ecosystems under acidic condition. The present study aimed to detect the amount of Al, Cu, Zn, Mn, Pb, Ni and Cd extracted by distilled water and ammonium acetate at pH 3.8, 4.8, 5.8, 6.8 and 7.8. The acute toxicities of water extracts were assessed by two bioassays. Results showed that concentrations of heavy metals, especially Al, were the highest in extracts from Al-contaminated soil extracted with NH₄OAc, followed by Al-waste. The control displayed relatively low levels of metals. More heavy metals were extracted at acidic pH than at neutral pH. Distilled water extracts exhibited lower levels of metals than those extracted with NH₄OAc. The first bioassay, in terms of seed germination and root elongation of B. parachinensis, indicated that the germination rates were seriously retarded by Al-waste even at neutral pH. Less than 25% seeds were germinated in Al-waste and the toxic effect was more obvious at pH 3.8. Root growth in Al-waste and Al-soil was slower than in the control and no seedling in Al-waste had roots longer than 2 cm at the end of this study. In the second bioassay, the photosynthetic rate of Chlorella pyrenoidosa was significantly inhibited by Al-waste when compared with the control, although the pH effect was not clear. This study revealed that the metal availability was pH dependent and their toxicity could be rapidly assessed by two simple bioassays.     

Conditions for the extraction of DNA and RNA from tobacco pollen by sodium chloride and perchloric acid

Studying the influence of the pH of 10% NaCl solutions used for the extraction of RNA and DNA on the yield of both nucleic acids, the maxima of pH were found at which both types of nucleic acids pass into extracts better than at neutral pH and do not remain in residues of the experimental material.The perchloric acid extraction temperature was also studied for obtaining the hydrolysate of nucleic acids from the trichloroacetic acid precipitate of sodium chloride extracts differing by 5 °C within the range of 35 °C to 90 °C and it was found that in this wide range almost the same amount of RNA is extracted by the method used. However, at a lower temperature, some DNA remained in the extracted residue of the trichloroacetic acid precipitate of sodium chloride extracts.     

Partial Purification and Some Properties of Polyphenoloxidases from Sago Palm

Two polyphenoloxidases (PPO I and PPO III, EC 1.10.3.1) were extracted and partially purified from sago palm pith by hydroxylapatite chromatography, DEAE-cellulose chromatography and gel filtration. Both purified isozymes gave a single activity band on polyacrylamide gel electrophoresis. The molecular weights of both enzymes were estimated to be 40,000. They had the same pH optima of 6.5 but different temperature optima, 35°C for PPO I and 45°C for PPO III. PPO I was stable at neutral to alkaline pH and PPO III at acidic pH. PPO III was somewhat more stable than PPO I when incubated at various temperatures for 15 min. PPO I and PPO III oxidized well DL-epicatechin and d-catechin, respectively. Both enzymes were strongly inhibited by KCN, Na-diethyldithiocarbamate and NaHSO₃.     

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

 Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [³H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins. Received: 5 November 1999 / Accepted: 18 January 2000     

The Separation of Pectic Polymers from Apple Fruit Tissue 3

A column chromatographic system utilizing diethylaminoethyl(DEAE) cellulose equilibrated with phosphate buffer at pH 6.5has been found satisfactory for the separation of pectic polysaccharidesfrom apple fruit tissue. Increasing phosphate concentrationseluted in order from a sample of Bramley Seedling whole pectin,a neutral arabinan-galactan, a polyuronide and a ‘ poly-aldo-uronide’. Similar components were found in extracts from Cox's OrangePippin tissue. The pectic fraction soluble in neutral bufferat 20 °C was found to contain largely polyuronide. Sodiumhexametaphosphate at 95 °C extracted a much larger proportionof poly-aldo-uronide but this component was partially degradedduring extraction. A proportion of polyuronide remaining afterthis extraction could be liberated only by markedly degradativeprocedures and was therefore not characterised.     

The solubilization of collagen and protein–polysaccharides from the developing cartilage of lathyrytic chicks

1. The solubilization of collagen and protein-polysaccharides from the developing cartilage of normal and lathyritic chicks was studied by using mild extraction procedures. One-third of the protein-polysaccharides could be solubilized in salt solutions at neutral pH from normal cartilage, whereas 95-100% could be extracted from the cartilage of animals that were severely lathyritic. Likewise, whereas in normal animals the collagen of cartilage was essentially insoluble in salt solutions at neutral pH, in lathyritic animals it was almost completely soluble. 2. The increased solubility of the collagen of cartilage from lathyritic animals enabled sufficient material to be collected so that the pure alpha1 chains of the collagen were isolated by repeated reconstitution, precipitation and CM-cellulose column chromatography. The purified alpha1 component was characterized by its relatively high content of hydroxylysine (14 residues/1000 amino acids). 3. About 37% of the collagen from the cartilage of normal chick embryos could be extracted as the gelatin at pH7.4 in lithium chloride solution. This was accompanied by the extraction of approx. 14% of the protein-polysaccharide content. 4. The protein-polysaccharides and the collagen from normal animals could be extracted from the cartilage relatively independently of one another under mild conditions. These same components obtained from lathyritic animals easily separated from one another after solubilization. This provided evidence that the two components are probably not covalently cross-linked. 5. The collagen of cartilage extracted as a gelatin from normal animals contained a high proportion of alpha chains compared with beta dimers, similar to the lathyritic collagen of cartilage and other tissues, and similar to the gelatin extracted from normal chick bone.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Hyaluronic acid in cartilage and proteoglycan aggregation

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.     

Recombinant cathepsin E has no proteolytic activity at neutral pH

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.     

Multiple carbohydrate-cleaving specificities in human acidic and neutral glycosidases.

The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.     

Neocarzinostatin: spectral characterization and separation of a non-protein chromophore.

Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.     

Biochemical studies on an acidophilic, thermophilic bacterium, Bacillus acidocaldarius: isolation of bacteria, intracellular pH, and stabilities of biopolymers.

Acidophilic, thermophilic bacteria were isolated from Japanese acidic hot springs. They were spore-forming rods, identified as Bacillus acidocaldarius. DNA extracted from these acido-thermophiles showed no abnormality in chemical structure; it was instantly denatured and gradually decomposed giving rise to apurinic acid in a hot acid environment milder than the optimal conditions for the growth of the acido-thermophiles. Glyceraldehyde-3-phosphate dehydrogenase extracted from B. acidocaldarius was not active at pH 5 or less, and was resistant to heat at neutral but not acid pH. The intracellular pH was computed to be neutral by using dimethyl-2,4-oxazolidinedione. When uncouplers or inhibitors of respiration were added to the cells suspended in hot acid solution, the estimated pH was not changed and glyceraldehyde-3-phosphate dehydrogenase in the cells was not denatured. These results suggest that the cytoplasm of B. acidocaldarius is a hot neutral environment, and that a pH gradient across the cell envelope can be maintained even when oxidative phosphorylation or respiration is inhibited.     

Bacterial action of fertilization envelope extract from eggs of the fish Cyprinus carpio and Plecoglossus altivelis

The vitelline envelope (VE) and fertilization envelope (FE) in eggs of the fish Cyprinus carpio and Plecoglossus altivelis were purified by homogenization of eggs or embryos in 5 mM Tris-HCl buffer, pH 7.0, containing 2 mM ethylenediamine tetraacetic acid disodium salt (EDTA), except for processing of VEs in Plecoglossus eggs, and by repeated washing wih the same buffer. To extract the outermost layer material, the purified VEs and FEs were processed overnight at 4 degrees C in 5 mM Tris-HCl buffer, pH 7.0, containing 8 mM 2-mercaptoethanol, 2 mM EDTA, 0.3 M alpha-lactose, 0.3 M glucose, and 0.9% NaCl. Since extraction of the outermost layer of the VEs of Cyprinus eggs in this solution was found to be ultrastructurally incomplete, further sonication in the same buffer was necessary. The solution extracted from purified VEs or FEs was dialyzed against 5 mM Tris-HCl buffer, pH 7.0, followed by lyophilization. The extracts from the FEs from both fish species contained two kinds of lectins, one agglutinated human B-type erythrocytes and the other nonspecifically agglutinated fish spermatozoa, and both extracts had a strong bactericidal effect on Vibrio anguillarum that was isolated from diseased cultured fish, but not on Aeromonas hydrophila and Escherichia coli. The extracts of purified VEs from eggs of both fish had no bactericidal effect on the bacteria examined, nor any agglutination effect on human erythrocytes and fish spermatozoa.     

Purification of rat renal renin from crude kidney extracts by diaminohexamethylene-Sepharose chromatography

Renin from rat kidney extracts was adsorbed to diaminohexamethylene-sepharose columns at extremely low ionic strength and neutral pH. Renin was retarded while the column was developed in 1 mM sodiumpyrophosphate and extraneous proteins were removed. Elution of renin was performed using a linear gradient of sodiumpyrophosphate, 1 – 17 mM at pH 6.8. Renin was purified in a yield up to approx. 60 per cent of the applied activity and a purification factor between 5 – 122 depending on the specific activity of the applied sample. The specific activity after this single chromatography of crude rat kidney homogenate on diaminohexamethylene-sepharose showed a median of 11.3 Goldblatt units per mg protein in a range of 5.3 – 42.0 Goldblatt units per mg protein. The renin binding capacity of the column was 1 Goldblatt unit per ml wet gel. The purified renin was subjected to G-100 Sephadex chromatography demonstrating two molecular weight forms of 44000 and 50000 dalton. Polyacrylamide gel electrophoresis demonstrated three separate fractions of renin.     

A rapid head-space method for ethyl alcohol determination in saliva samples

At neutral pH, tetraphenylboron dissolved in 3-heptanone extracts acetylcholine but not acetylcarnitine from aqucous media. At acid pH, both acetylcholine and acetylcarnitine are extracted. A pH curve of the efficiency of extraction of acetylcholine and acetylearnitine is given and the method of chemical synthesis of [1-acetyl-¹⁴C]carnitine is deseribed.     

Activity,conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles

The adsorption of a recombinant cutinase from Fusarium solani pisi onto the surface of 100 nm diameter poly(methyl methacrylate) (PMMA) latex particles was evaluated. Adsorption of cutinase is a fast process since more than 70% of protein molecules are adsorbed onto PMMA at time zero of experiment, irrespective of the tested conditions. A Langmuir-type model fitted both protein and enzyme activity isotherms at 25 degrees C. Gamma(max) increased from 1.1 to 1.7 mg m(-2) and U(max) increased from 365 to 982 U m(-2) as the pH was raised from 4.5 to 9.2, respectively. A decrease (up to 50%) in specific activity retention was observed at acidic pH values (pH 4.5 and 5.2) while almost no inactivation (eta(act) congruent with 87-94%) was detected upon adsorption at pH 7.0 and 9.2. Concomitantly, far-UV circular dichroism (CD) spectra evidenced a reduction in the alpha-helical content of adsorbed protein at acidic pH values while at neutral and alkaline pH the secondary structure of adsorbed cutinase was similar to that of native protein. Fluorescence anisotropy decays showed the release of some constraints to the local motion of the Trp69 upon protein adsorption at pH 8.0, probably due to the disruption of the tryptophan-alanine hydrogen bond when the tryptophan interacts with the PMMA surface. Structural data associated with activity measurements at pH 7.0 and 9.2 showed that cutinase adsorbs onto PMMA particles in an end-on orientation with active site exposed to solvent and full integrity of cutinase secondary structure. Hydrophobic interactions are likely the major contribution to the adsorption mechanism at neutral and alkaline pH values, and a higher amount of protein is adsorbed to PMMA particles with increasing temperature at pH 9.2. The maximum adsorption increased from 88 to 140 mg cutinase per g PMMA with temperature raising from 25 to 50 degrees C, at pH 9.2.