Interference of biogenic amines with the measurement of proteins using bicinchoninic acid

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.     

Quantitative determination of zwitterionic detergents using salt-induced phase separation of Triton X-100

Zwitterionic detergents interfere with the salt-induced phase separation for nonionic detergents in a concentration-dependent manner by shifting the normal cloud point of nonionic detergents to a higher ionic strength at room temperature. This phenomenon was used to determine the concentration of the zwitterionic detergents CHAPS, CHAPSO, and sulfobetaine SB-12 in solution by titration with ammonium sulfate in the presence of Triton X-100. Among the ionic detergents tested, the method was only applicable to sodium cholate. The assay can be used to control the removal of zwitterionic detergents during the reconstitution of membrane proteins in liposomes. However, it cannot be used to determine the specific binding of zwitterionic detergents to highly diluted, pure membrane proteins because of the limited sensitivity. Neither proteins nor phospholipids interfered with this method at concentrations up to 20 mg/ml of test solution (human serum albumin) or 10 mg/ml (phospholipids), respectively. Since the assay is based on the competition between salts and nonionic detergents for water molecules, it is important to equalize the ionic strength of samples and calibration standards.     

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.     

家用洗涤剂对红剑鱼、孔雀鱼和食蚊鱼的急性毒性实验观察

目的 研究4种不同产地的家用洗涤剂对红剑鱼、孔雀鱼和食蚊鱼的急性毒性影响。方法 用水毒理学方法测定它们对3种鱼类的24hLC50,并将洗涤剂溶液存放15d和30d后测定其对鱼类的急性毒性。结果 在远低于日常使用量的浓度下,4种家用洗涤剂均对红剑鱼、孔雀鱼和食蚊鱼有急性毒性,24h LC50值在20mg／L～55mg／L;家用洗涤剂溶液存放一段时间后对鱼类的急性毒性作用无明显降低。结论 含有大量家用洗涤剂的生活污水排放到自然水体后将对鱼类产生持续的有害影响。     

Method for Lyophilizing Brain Proteolipid Preparation That Increases Subsequent Solubilization by Detergent

Abstract: A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2 mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X-100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X-100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed.     

Characterization of UDP-galactosyl:asialo-mucin transferase activity in the Golgi system of rat liver.

UDPgalactosyltransferase activity (UDPgalactose:mucopolysaccharide galactosyltransferase, EC 2.4.1.74) was measured in a well-characterized fraction of Golgi membranes in the presence of UDPgalactose and exogenous acceptor sites. Substrate saturation for 0.05 mg Golgi protein was achieved at a concentration of 4.6 mM UDPgalactose. Desialylated mucin proved to be the most suitable acceptor protein. Access to galactose acceptor sites was not rate limiting for the reaction when 20 mg of asialo-mucin/ml of incubation mixture was used. With these concentrations of substrates the use of nucleotides to inhibit pyrophosphatases and of detergents to perturb the membrane structure was not necessary and proved, in fact, to be inhibitory to galactose transfer. UDPgalactosyl:asialo-mucin transferase activity in Golgi membranes was 230 nmol galactose transferred/mg Golgi protein per 30 min.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Disinfectant effect of Persteril in combination with detergents

In laboratory conditions, the microbicidal effect, pH and changes in the content of peracetic acid and hydrogen peroxide were tested in Persteril at concentrations of 5 ml/l and 0.5 ml/l as well as in mixtures of these Persteril solutions with the detergents Jar, Pur, Hit, Corona, Sapon, Rekord and Universal. The efficiency and stability of Persteril solution in combination with the detergents were similar to those of Persteril aqueous solution. The tested mixtures ensured satisfactory bactericidal effect after 19-day storage. The sporicidal effect could be guaranteed during 5 days only at a concentration of 5 ml/l and provided disinfection was carried out by submerging. The above mixtures of Persteril and detergents have been recommended for one-stage disinfection in all types of medical facilities requiring simultaneous disinfection and washing.     

Potential Use of Surface-active Agents for Controlling Mycoplasma Contamination in Animal Cell Cultures

Seven nonionic detergents, which were determined to be relatively nontoxic to selected animal cell cultures, were tested for their lethal effect on the GDL strain of Mycoplasma hyorhinis. Of the seven detergents tested, five were found to cause complete lysis of the organism in vitro within 24 hr at 37 C. These detergents included Triton WR-1339 and Tweens 20, 40, 60, and 80. When different concentrations of the detergents were tested, Tween 80 was found to be the most effective and Triton WR-1339 the least effective in lysing the mycoplasmata. These same five detergents were used to treat a rat nephroma cell line which was chronically infected with the GDL strain. The mycoplasmata were eliminated from those cultures treated with Triton but they persisted in cultures exposed to the Tween compounds. The Triton-treated cells remained free from infection over a 7-month period, as determined both by cultural methods and fluorescent-antibody staining. The "cure" was effected by treating the cells for either 48 hr with maintenance media containing 1 mg of Triton per ml or for 96 hr with a concentration of 500 mug/ml. Triton was also effective in eliminating the GDL, strain from experimentally infected rat embryo cells after a 48-hr treatment with a concentration of 1 mg/ml. Four other species of Mycoplasma, which were completely lysed by Triton in vitro, were not eliminated from experimentally infected cells by a single treatment with Triton, although the severity of the infection was apparently reduced.     

Responses to the lowering of magnesium and calcium concentrations in the cerebrospinal fluid of unanesthetized sheep.

A technique for ventriculolumbar perfusion of the cerebrospinal fluid space has been used to study the neuromuscular effects of low concentrations of magnesium and calcium in the cerebrospinal fluid of conscious sheep. Perfusion with synthetic cerebrospinal fluid solutions containing less than 0-6 mg magnesium/100 ml produced episodes of tetany which were abolished by perfusion with a solution of normal magnesium concentration. This suggests that the low cerebrospinal fluid magnesium concentrations reported in cases of hypomagneseamic tetany may result in changes within the central nervous system that could produce the nervous signs. Perfusates with a calcium concentration below 2-0 mg/100 ml caused hyperpnoea and continuous muscle tremors. Magnesium (0-6 mg/100 ml) and calcium (2-0 mg/100 ml) perfused simultaneously acted synergistically to produce signs characteristic of low levels of each of the ions.     

Sequential use of detergents for solubilization and reconstitution of a membrane ion transporter.

Solubilization and reconstitution of the cardiac sarcolemmal Na+/Ca2+ exchanger by use of the anionic detergent cholate and its application for reconstitution of the exchanger following solubilization with zwitterionic or nonionic detergents is described. Solubilization and reconstitution with cholate provided a 32.6-fold enrichment of Na+/Ca2+ exchange activity over sarcolemmal vesicles (5.2 to 170 nmol/mg/s) with 202% recovery of total activity. In combination with asolectin, the cholate dilution technique (H. Miyamoto and E. Racker, J. Biol. Chem. 255, 2656, 1980) offers a rapid and simple means for reconstitution and provides good recovery of total and specific Na+/Ca2+ exchange activity. However, the use of anionic detergents for solubilization precludes the use of certain chromatographic procedures for protein purification. Conversely, nonionic and zwitterionic detergents permit effective use of available chromatographic techniques, but can be troublesome during reconstitution. We have combined the advantages of solubilization with nonionic and zwitterionic detergents with the advantages of reconstitution by cholate dilution. Reconstitution of the exchanger, after solubilization with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (Chaps) or n-octyl-beta-D-glucoside, was accomplished by the addition of a cholate/asolectin medium followed by dilution. Na+/Ca2+ exchange activity was enriched 30.7-fold with 196% recovery with Chaps and 34.1-fold with 204% recovery with n-octyl-beta-D-glucoside. The presence of Chaps was found to shift the optimal asolectin concentration for reconstitution from 15 mg/ml (cholate alone) to 25 mg/ml. In addition, pelleting of proteoliposomes subsequent to reconstitution resulted in greatest recovery of total activity when volumes were kept below 1.0 ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

Toxicity of synthetic detergents to fish and aquatic invertebrates

Synthetic detergents are reported to be acutely toxic to fish in concentrations between 0.4 and 40 mg/1. Factors affecting toxicity include the molecular structure of the detergent, water hardness, temperature and dissolved oxygen concentration; the age and species of the test fish, and acclimation to low concentrations of detergent. Some of these factors appear to be of only limited importance. Gill damage is the most obvious acute toxic effect; the immediate cause of death may be asphyxiation, but detergents may also be toxic internally. Lethal effects not related to gill damage have not been investigated. Sublethal effects include retardation of growth, alteration of feeding behaviour and inhibition of chemoreceptor organs. Low levels of detergents may also increase the uptake of other pollutants. Invertebrates, especially in their juvenile stages, are extremely sensitive to detergents: concentrations below 0.1 mg/1 interfere with growth and development in some species. The interactions between detergents and proteins, and their influence on membrane permeability may be the basis of the biological action of detergents. Detergents in natural waters are usually partially degraded, and a maximum permissible concentration of 0.5 mg/1 would probably be harmless under most conditions.     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase

Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.     

A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay

A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of 125I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of 125I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with 125I-labeled alpha-bungarotoxin and antigens with 125I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the 125I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.     

Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor.

Oxytocin receptors were measured in myometrium and intercaruncular endometrium of cows during pregnancy and parturition. Concentrations of estradiol-17 beta, estrone, and progesterone in peripheral blood were also measured. Receptor concentrations in the endometrium rose almost 200-fold from Day 20 to term (p < 0.0001, ANOVA), from 40 +/- 11 to 7300 +/- 1430 fmol/mg protein. Myometrial receptor concentrations increased 10-fold from 180 +/- 36 fmol/mg on Day 20 to 1850 +/- 360 fmol/mg protein at term (p < 0.0001, ANOVA). During labor, endometrial receptors (6600 +/- 1300 fmol/mg) remained at prelabor values, whereas myometrial receptor concentrations had decreased to 1190 +/- 316 fmol/mg (not significant) and declined further postpartum. Plasma concentrations of progesterone declined from 4-5 ng/ml to about 2 ng/ml between Days 250 and 282 and dropped to < 0.2 ng/ml shortly before delivery. Plasma concentrations of estrone and estradiol-17 beta were below 10-20 pg/ml until Day 230. Estrone concentrations were significantly (p < 0.05) increased by Day 250 and estradiol-17 beta by Day 270, and then both rose rapidly. During labor, plasma estrone was 1135 +/- 245 pg/ml and plasma estradiol-17 beta was 226 +/- 131 pg/ml. The molar ratio of estrone and estradiol-17 beta to progesterone rose from less than 0.01 to 4.4 during labor, and was correlated with oxytocin receptor concentrations in endometrium (r = 0.5160, p < 0.001), but not those in myometrium (r = 0.0122). The regulation of oxytocin receptors by ovarian hormones in the two tissues may therefore differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of albumin on the in vitro conjugation of bilirubin by rat liver microsomes

These studies were carried out to determine whether bovine serum albumin (BSA), which is usually included in the incubation mixture for the in vitro determination of bilirubin-UDP-glucuronyl transferase (GT) activity, affects GT activity. Using bilirubin as substrate, addition of BSA to the enzyme reaction mixture at concentrations varying from 2 to 30 mg/ml resulted in a dose-related inhibition of "native" GT activity of rat liver microsomes. When detergent-activated enzyme was employed, increasing concentrations of BSA also required higher concentrations of deoxycholate, digitonin, or Triton X-100 to produce maximal bilirubin conjugation. Low BSA concentrations (2 mg/ml) prevented enzyme activation by both detergents and UDP-N-acetyl glucosamine. When BSA was omitted and bilirubin dissolved in dimethyl sulfoxide, UDP-N-acetyl glucosamine failed to enhance GT activity, and activation by detergents was only 15-25% of that observed in the presence of optimal concentrations of BSA. When rat albumin was substituted for BSA, a similar dose-related inhibition of in vitro bilirubin conjugation by untreated microsomes was observed, although at any given albumin concentration, GT activity was lower with rat than with bovine albumin. Additionally, both detergents and UDP-N-acetyl glucosamine produced similar GT activation regardless of the rat albumin concentration. Finally, these effects of BSA and rat albumin could not be reproduced when beta-lactoglobulin was employed and/or when p-nitrophenol was the acceptor substrate of GT. These findings indicate that albumin, in particular BSA, profoundly and selectively influences the in vitro activity of microsomal GT toward bilirubin as the acceptor substrate.