Comparison of spectrolyser device measurements with standard analysis of wastewater samples in Novi Sad, Serbia.

On-line monitoring was performed using spectrolyser equipment, coupled with laboratory analysis for samples collected from wastewater discharge in the city of Novi Sad, Serbia, during first 24 h of three and 48 h of six monitoring campaigns from December of 2012 to April of 2013. Significant correlation with R(2) > 0.9 was observed between laboratory analysis and spectrolyser measurements for chemical oxygen demand (COD) and biological oxygen demand (BOD) concentrations. COD/BOD5 ratio in combined industrial and municipal wastewater ranged from 1.2 to 2.0 indicating the presence of biodegradable organic matter which could be easily removed using aeration treatment process. Micro/trace element and/or heavy metals in wastewater samples were within the limits as per the standard prescribed for wastewater, and should not pose any serious hazard risk. However BOD, COD, ammonia and total phosphorus concentrations were measured above the limit value according to Serbian and EU legislation and should be reduced before discharging wastewater directly into the Danube River.

[Effects of oral administration of bifidobacteria on intestinal microflora in premature and newborn infants]. / Wirkungen oral verabreichter Bifidobakterien auf die intestinale Mikroflora von Früh- und Neugeborenen.

In a prospective, randomised study the effects of orally administered bifidobacteria on the intestinal microflora were investigated in 100 preterm and term neonates under intensive care conditions during the first 21 days of life. The 50 infants (group with bifidobacteria) received lyophilized bifidobacteria (Töpfer Bifidus) via nasogastral tube with an initial dosage of 3 times daily 1.25 x 10(8) bifidobacteria on day 2 of life and a daily dosage of 6 times 1.25 x 10(8) bifidobacteria on day 3 until day 21 of life. The other 50 infants (control group) did not receive bifidobacteria. The preterm and term neonates were fed either with pasteurized mother's milk or milk from healthy female donors (n = 79) or with an infant formula (Alfaré, n = 13) or initially with Alfaré and thereafter with mother's milk (n = 8). The intestinal microflora of preterm and term neonates under intensive care conditions could be influenced by the oral administration of bifidobacteria. The administration of bifidobacteria resulted in the group of inoculated infants in a significantly earlier colonization of bifidobacteria (8.1 +/- 3.9 days of life) than in the control group (11.3 +/- 4.7 days of life). On day 7 a bifidobacterial dominance (> 90% of the intestinal microflora) could be found in 26% of infants with inoculation of bifidobacteria and only in 2% of the control group (p < 0.001). These significant differences could be shown until day 21 of life. A difference in septicemia frequency between the two groups could not be demonstrated. At the beginning of the infection a bifidobacterial dominance was found in only one of 23 cases of septicemia.

Vascular malformations of the upper limb: a review of 270 patients.

Vascular malformations of the upper limb were once thought to be impossible to properly diagnose and treat. We reviewed our experience with these malformations of the upper limb in 270 patients seen over a 28-year period. These anomalies were slightly more common in females than males (ratio, 1.5:1.0). The malformations were categorized as either slow flow (venous, n = 125; lymphatic, n = 47; capillary, n = 32; combined, n = 33) or fast flow (arterial, n = 33). Three categories of fast-flow malformations were identified and designated as types A, B, and C. Over 90% of these lesions could be properly diagnosed by their appearance and growth pattern within the first 2 years of life. Additional radiographic studies were used to confirm this diagnosis and to define specific characteristics. Magnetic resonance imaging with and without contrast best demonstrated site, size, flow characteristics, and involvement of contiguous structures for all types of malformations. Algorithms for treatment of both slow-flow and fast-flow anomalies are presented. Two hundred sixty surgical resections were performed in 141 patients, including 24 of 33 fast-flow anomalies. Preoperative angiographic assessment, with magnified views, was an important preoperative adjunct before any well-planned resection of fast-flow arteriovenous malformations. The surgical strategy in all groups was to thoroughly extirpate the malformation, with preservation of nerves, tendons, joints, and uninvolved muscle, and microvascular revascularization and skin replacement as required. Resections were always restricted to well-defined regions and often completed in stages. Symptomatic slow-flow malformations and types A and B fast-flow anomalies were resected without major sequelae. Type C arterial anomalies, diffuse, pulsating lesions with distal vascular steal, and involvement of all tissues, including bone, progressed clinically and resulted in amputation in 10 of 14 patients. The complication rate was 22% for slow-flow lesions and 28% for fast-flow lesions.

Characteristics of cartilage engineered from human pediatric auricular cartilage.

In the repair of cartilage defects, autologous tissue offers the advantage of lasting biocompatibility. The ability of bovine chondrocytes isolated from hyaline cartilage to generate tissue-engineered cartilage in a predetermined shape, such as a human ear, has been demonstrated; however, the potential of chondrocytes isolated from human elastic cartilage remains unknown. In this study, the authors examined the multiplication characteristics of human auricular chondrocytes and the ability of these cells to generate new elastic cartilage as a function of the length of time they are maintained in vitro. Human auricular cartilage, harvested from patients 5 to 17 years of age, was digested in collagenase, and the chondrocytes were isolated and cultured in vitro for up to 12 weeks. Cells were trypsinized, counted, and passaged every 2 weeks. Chondrocyte-polymer (polyglycolic acid) constructs were created at each passage and then implanted into athymic mice for 8 weeks. The ability of the cells to multiply in vitro and their ability to generate new cartilage as a function of the time they had been maintained in vitro were studied. A total of 31 experimental constructs from 12 patients were implanted and compared with a control group of constructs without chondrocytes. In parallel, a representative sample of cells was evaluated to determine the presence of collagen. The doubling rate of human auricular chondrocytes in vitro remained constant within the population studied. New tissue developed in 22 of 31 experimental implants. This tissue demonstrated the physical characteristics of auricular cartilage on gross inspection. Histologically, specimens exhibited dense cellularity and lacunae-containing cells embedded in a basophilic matrix. The specimens resembled immature cartilage and were partially devoid of the synthetic material of which the construct had been composed. Analyses for collagen, proteoglycans, and elastin were consistent with elastic cartilage. No cartilage was detected in the control implants. Human auricular chondrocytes multiply well in vitro and possess the ability to form new cartilage when seeded onto a three-dimensional scaffold. These growth characteristics might some day enable chondrocytes isolated from a small auricular biopsy to be expanded in vitro to generate a large, custom-shaped, autologous graft for clinical reconstruction of a cartilage defect, such as for congenital microtia.

Improved axon diameter and myelin sheath thickness in facial nerve cable grafts wrapped in temporoparietal fascial flaps.

Injury to the facial nerve in the temporal bone presents a challenge to the recovery of nerve function, in that the fallopian canal in which it lies is poorly vascularized. This study was designed to determine if wrapping an intratemporal facial nerve defect repaired with a cable graft with a well-vascularized temporoparietal fascial (TPF) flap would improve facial nerve regeneration. To evaluate this question, a defect was created in the intratemporal left facial nerve of 10 rabbits. All nerves were repaired using cable grafts. In 5 animals, the nerve graft was wrapped with temporoparietal fascia, whereas in the other 5 rabbits it was not. Three additional animals underwent exposure only. The contralateral nerve served as a control in all animals. Quantitative analysis of the nerve graft 12 weeks after repair revealed greater recovery of original fiber diameter and myelin sheath thickness in TPF flap-wrapped repairs. Histological evidence of improved neural regeneration and functional nerve recovery was also seen in the repairs where the TPF flap was utilized. Nerve conduction and electromyographic studies of the cable-grafted nerve at 6 and 12 weeks were equivocal, however.

Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte-macrophage colony-stimulating factor for hematologic support after high-dose intensification for breast cancer.

High-dose therapy with autologous marrow support results in durable complete remissions in selected patients with relapsed lymphoma and leukemia who cannot be cured with conventional dose therapy. However, substantial morbidity and mortality result from the 3- to 6-week period of marrow aplasia until the reinfused marrow recovers adequate hematopoietic function. Hematopoietic growth factors, particularly used after chemotherapy, can increase the number of peripheral blood progenitor cells (PBPCs) present in systemic circulation. The reinfusion of PBPCs with marrow has recently been reported to reduce the time to recovery of adequate marrow function. This study was designed to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized PBPCs alone (without marrow) would result in rapid and reliable hematopoietic reconstitution. Sixteen patients with metastatic breast cancer were treated with four cycles of doxorubicin, 5-fluorouracil, and methotrexate (AFM induction). Patients responding after the first two cycles were administered GM-CSF after the third and fourth cycles to recruit PBPCs for collection by two leukapheresis per cycle. These PBPCs were reinfused as the sole source of hematopoietic support after high doses of cyclophosphamide, thiotepa, and carboplatin. No marrow or hematopoietic cytokines were used after progenitor cell reinfusion. Granulocytes greater than or equal to 500/microL was observed on a median of day 14 (range, 8 to 57). Transfusion independence of platelets greater than or equal to 20,000/microL occurred on a median day of 12 (range, 8 to 134). However, three patients required the use of a reserve marrow for slow platelet engraftment. In retrospect, these patients were characterized by poor baseline bone marrow cellularity and poor platelet recovery after AFM induction therapy. When compared with 29 historical control patients who had received the same high-dose intensification chemotherapy using autologous marrow support, time to engraftment, antibiotic days, transfusion requirements, and lengths of hospital stay were all significantly improved for the patients receiving PBPCs. Thus, autologous PBPCs can be efficiently collected during mobilization by chemotherapy and GM-CSF and are an attractive alternative to marrow for hematopoietic support after high-dose therapy. The enhanced speed of recovery may reduce the morbidity, mortality, and cost of high-dose treatment. Furthermore, PBPC support may enhance the effectiveness of high-dose therapy by facilitating multiple courses of therapy.

Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.

[Comparison of results of daily blood pressure self-monitoring and general practice monitoring within the scope of a study assessing treatment of hypertension with quinapril]. / Vergleich der Ergebnisse von täglichen Blutdruckselbstmessungen und Praxismessungen im Rahmen einer Studie zur Behandlung der Hypertonie mit Quinapril (Accupro).

24 hour blood pressure monitoring is a well established method in the field of antihypertensive research. Patients self recorded blood pressure values are an additional option to overcome the disadvantages of casual office readings--however they are not frequently used within intervention trials. To prove the usefulness of selfrecordings in clinical trials we investigated both selfrecordings taken twice a day and casual readings within intervals of 1 to 3 weeks, in this study on the efficacy and tolerability of the ACE-inhibitor Accupro. 108 hypertensive patients (grade WHO I to II) were included in this trial for ten weeks. Although blood pressures were measured by the patients using sphygmomanometers of the same type and the physicians, decisions to treat or to increase dosage were based on the patients' recordings only. Accupro was dispensed according to the package leaflet at a daily dosage of 5 mg up to 40 mg. In case of failing response to monotherapy, Accupro was combined with Diltiazem or with a diuretic. 7 patients discontinued the treatment due to mild adverse events, one did not cooperate. 82 of the remaining patients were treated effectively with Accupro monotherapy--60 (73%) got one dose daily, 22 (27%) 2 doses per day,--and in 18 patients a drug combination was required. Therapeutic response (RRd < or = 90 mm Hg) was gained within 86 of the 100 evaluable patients according to the doctors' and 83 according to the patients' records. In this respect the two methods used gave comparable overall results. This somewhat surprising fact is due to the design of the study, because treatment decisions were based on the selfrecordings only. Clinical trials based on selfrecordings are in some points preferable to casual office readings: As patients being normotensive at home should not be included into an interventional study, a change of dosage within this group is avoided. Additionally the compliance of a cooperative patient taking his blood pressure twice daily is at a high level. Measurements of each single patient may be evaluated statistically by time series-analysis regarding longterm distribution of blood pressure-values. Taking the means of selfrecordings over adequate time-intervals eliminates the influence of "outliers" (occasionally extremely high or low values) and also reduces the standard deviation compared to that of the casual readings. Research work based on self recordings provides more information and therefore more security for treatment decisions.

GM-CSF potentiated peripheral blood progenitor cell (PBPC) collection with or without bone marrow as hematologic support of high-dose chemotherapy: two protocols.

High-dose chemotherapy with autologous bone marrow support (ABMS) achieves prolonged relapse-free survival in relapsed lymphomas and leukemias and has provided durable complete responses in certain solid tumors. The principal morbidity and mortality result from the infectious and bleeding complications during the 3-4 week aplasia until the bone marrow autograft can recover. Hematopoietic growth factors, alone or used after chemotherapy, increase the number of circulating progenitor cells in the peripheral blood compartment. In one trial, 12 patients with solid tumors were treated with high-dose chemotherapy and supported with both bone marrow and peripheral blood progenitor cells (PBPC) collected after GM-CSF administration. Reconstitution of bone marrow function occurred quickly (ANC greater than 500/microliters by day 17; platelet-transfusion independence by day 16), resulting in short hospital stays (median, 28 days). In a second study, 12 patients with metastatic breast cancer responding to induction chemotherapy (doxorubicin, 5-fluorouracil, and methotrexate) were given GM-CSF during induction to collect PBPCs during leukocyte recovery. These PBPCs were used as the sole hematopoietic support during high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin. Granulocyte and platelet reconstitution were extremely rapid (median, 14 and 12 days, respectively). When compared with 29 patients undergoing the same intensification therapy using ABMT as sole support, time to hematopoietic recovery, transfusion requirements, and duration of hospital stay were all significantly improved for the patients receiving PBPC. PBPC with or without marrow may enhance the safety, tolerance, and cost of high-dose therapy. Moreover, PBPC may render multiple course combination, high-dose therapy feasible.

Kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin as measured by small-angle neutron scattering.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into two half-molecular fragments was investigated at 21.0 degrees C by using small-angle neutron scattering. The relative change in molecular mass that occurs upon dissociation was monitored by recording the forward scattering of neutrons as a function of time. All these kinetic data can be explained by a reaction that is first-order with respect to the concentration of undissociated alpha 2-macroglobulin. The velocity constant is a function of urea concentration and it varies within wide limits. For instance, the half-life of the reaction at the lowest concentration of [2H]urea studied (2.70 M) is 328 h, whereas the same value at the highest concentration of [2H]urea (6.24 M) is only 8 min. Measurements were made both with [1H]urea in 1H2O and with [2H]urea in 99% 2H2O, and it was found that there is a pronounced kinetic isotope effect, i.e. the dissociation is 4 times faster in the 1H-containing medium as compared with the 2H-containing medium at the same molar concentration of urea. From the angular dependence of the neutron scattering it can be concluded that the dissociation is associated with a drastic change in structure. This is directly shown by the radius of gyration, which increases from about 7.4 nm immediately after the addition of urea up to about 9.4 nm when the protein is fully dissociated. A structural analysis shows that the scattering curve of urea-dissociated alpha 2-macroglobulin can best be explained by that of a Gaussian coil with a radius of gyration equal to 9.44 nm. These data indicate that the so-called non-covalent interaction of alpha 2-macroglobulin probably is more complicated than just a pure hydrophobic interaction. Finally, it is also shown that the dissociation is accompanied by a loss in trypsin-binding activity, which is directly related to the fraction of dissociated protein.

The H(+)-induced dissociation of human plasma alpha 2-macroglobulin. An investigation using small-angle neutron scattering and test of trypsin binding activity.

The dissociation of the tetrameric alpha 2-macroglobulin molecule into two half-molecular fragments, which occurs at pH less than 4.5, has been investigated using the small-angle neutron scattering method, and test of trypsin binding activity. Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid-base groups, one per monomeric subunit of alpha 2-macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H(+)-binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2-4.5, suggests that they are most probably carboxylate groups. From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein. Thus, at pH less than 4.5, there is a transition of alpha 2-macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.

Evaluation of monoclonal antibody DF3 conjugated with ricin as a specific immunotoxin for in vitro purging of human bone marrow.

DF3 is an IgG1 monoclonal antibody (MAb) generated against a Mr 350,000-400,000 glycoprotein expressed by approximately 80% of human breast cancers. We have coupled MAb DF3 to ricin. Purification of the immunotoxin (DF3-IT) was obtained by affinity and size exclusion chromatography. DF3 antigen-positive breast cancer cell lines (ZR-75-1, BT-20, and MCF-7) and DF3 antigen-negative lung cancer cell lines (A549 and CALU6) were tested for cytotoxicity with metabolic labeling and clonogenic assays. The cells were exposed for 3 h to different concentrations of DF3-IT, MAb DF3, ricin, and a combination of unconjugated MAb DF3 and ricin. In the presence of 100 mM lactose, DF3-IT specifically inhibited protein synthesis of lines expressing DF3 antigen on their cell surface. Moreover, clonogenic survival experiments demonstrated that DF3-IT at a concentration of 1 x 10(-9) M specifically kills 2.6-2.8 log of ZR-75-1 and BT-20 cells and 1.6 log of MCF-7 cells. At the same concentration, nonspecific toxicity of DF3-IT resulted in a 30% reduction of bone marrow granulocyte and macrophage colony formation. In bone marrow-purging experiments, tumor cells were mixed with an excess of bone marrow cells and treated with DF3-IT or ricin. Tumor cell clonogenic survival assays demonstrated that the presence of bone marrow cells had no detectable effect on activity or specificity of the DF3-IT. These results thus indicate that MAb DF3 is an effective vehicle to specifically deliver toxins to cancer cells which express DF3 antigen on their surface and that DF3-IT may be useful for in vitro purging of bone marrow.

Purging of small cell lung cancer-contaminated bone marrow by monoclonal antibodies and magnetic beads.

At diagnosis, most patients with small cell lung cancer (SCLC) have bone marrow involvement detected by immunological techniques. As preparative regimens become more effective in the elimination of systemic disease, bone marrow purging may become a significant intervention, even more so with the trend toward use of greater numbers of stem cells for reconstitution (such as peripheral blood stem cells). The optimal conditions for removal of SCLC cell lines from bone marrow mixtures with magnetic bead immunoconjugates are described. Metabolically labelled SCLC cells (SW2, OC2, NCI-H82) were incubated with mouse monoclonal antibodies reactive with SCLC membrane antigens (SM1, LAM2, LS1) alone or in combination. Magnetic beads conjugated with secondary goat anti-mouse-IgG or IgM antibody were added and the unpurged cells decanted off. Tumor removal was evaluated by residual radioactive counts in the decanted cell mixture as well as by clonogenic assay. The log removal was compared to controls performed without primary or secondary antibody. All assays were performed in triplicate. Bone marrow toxicity was similarly evaluated. In a simulation of bone marrow purging, a bead to total cell ratio of greater than or equal to 10:1 provided maximal tumor removal regardless of the degree of contamination. However, when the bead to total cell ratio dropped lower than 10:1, purging efficiency lessened despite maintenance of bead: tumor cell ratio of 100:1. A concentration of primary antibody ten-fold greater than saturation by indirect immunofluorescence provided optimal elimination. Using single exposures of antibody and beads, a mixture of all 3 MoAbs resulted in 2.4-2.6 log removal for SW2 and OC2. However, with NCI-H82, a cell line with low expression of these antibodies on the cell surface, maximal elimination was only 1.6 logs. Double bead exposure for 30 minutes each was more effective than single treatments for 30-60 minutes. No significant toxicity to bone marrow function was observed in parallel studies. Magnetic bead separation appears to be an efficient and nontoxic method to purge bone marrow of tumor contamination with SCLC.

Molecular organization of human plasma alpha 2-macroglobulin and its trypsin complexes. A small-angle x-ray scattering investigation.

The molecular organization of human plasma alpha 2-macroglobulin (alpha 2M), and its 1:1 and 1:2 trypsin complexes, have been investigated using the small-angle x-ray scattering method. All the experimental data can be explained by the same basic model, consisting of three oblate-shaped domains arranged in a sandwich-like structure. Each of the larger peripheral domains consists of two parallel elliptic cylinders associated side-by-side, whereas the smaller central domain consists of just one elliptic cylinder. In the native molecule the three domains are separated by regions of low protein density. Upon trypsin binding the dimensions of the four peripheral cylinders remain unchanged, but their positioning in space is reorganized so that the whole molecule becomes more compact. The model thus offers a plausible explanation for the mechanism of inactivating of the protease by entrapping it between the two larger domains. By comparing the shape and dimensions of the total molecule with those determined for the half-molecular fragment, obtained after reducing the intersubunit disulfide bonds, we propose that the fragment consists of just one of the peripheral domains plus half of the central domain. Different projections of the model are consistent with the electron micrographs of alpha 2M given in the literature. The model can also explain many of the physical and chemical properties recorded for alpha 2M and its protease complexes.

Solution structure of human plasma fibronectin as a function of NaCl concentration determined by small-angle X-ray scattering.

The structure of human plasma fibronectin in 50 mM Tris-HCl buffer, pH 7.4, containing varying concentrations of NaCl, has been investigated using the small-angle X-ray method. Below 0.3 M NaCl the overall structure of the molecule is disc-shaped; at 0 M NaCl the axial ratio of the disc is about 1:7 and between 0.1 M to 0.3 M it is slightly more asymmetric, with an axial ratio of 1:10. At about 0.3 M NaCl there is a reversible transition to a more open structure, and, from 0.3 M up to 1.1 M NaCl the small-angle X-ray data can be explained by models consisting of ensembles of flexible, non-overlapping, bead-chains generated by a Monte Carlo procedure. Within this concentration range there is a gradual increase in the stiffness of the chains, as well as a decrease in bead radius, which indicates that the molecule becomes more open when the NaCl concentration is increased. The transition to a more open structure is also demonstrated by the average radius of gyration which increases gradually from 8.26 nm at 0 M NaCl to 8.75 nm at physiological or near-physiological conditions, and up to 16.2 nm at 1.1 M NaCl.

Solution structure of human plasma fibronectin using small-angle X-ray and neutron scattering at physiological pH and ionic strength.

Human plasma fibronectin has been investigated at physiological pH and ionic strength, by using small-angle X-ray and neutron scattering techniques. The results indicate that the molecule is disc shaped with an axial ratio of about 1:10. In fact, an ellipsoid of revolution with semiaxes a = 1.44 nm and b = c = 13.8 nm is in agreement with the experimental scattering data, and can also fully explain the rather extreme hydrodynamic parameters reported for fibronectin. The X-ray data gave a radius of gyration of 8.9 nm and a molecular weight of 510,000, whereas the neutron data gave slightly larger values, 9.5 nm and 530,000, respectively. From the volume of the best fitting ellipsoid we obtain a degree of hydration of 0.61 g H2O/g protein (dry weight). Neutron data, recorded at different D2O concentrations in the solvent, gave a match point of 43% D2O, which indicates that approximately 80% of the hydrogens bound to oxygen and nitrogen are exchangeable.

Dodecylsulfate-induced dissociation of human alpha 2-macroglobulin. An investigation using small-angle neutron scattering and the equilibrium dialysis technique.

The dodecylsulfate-induced dissociation of the tetrameric alpha 2-macroglobulin molecule from human plasma has been investigated by the small-angle neutron scattering (SANS) method. The great advantage with the SANS method is that, by using deuterated dodecylsulfate, and contrast variation by changing the D2O/H2O ratio of the solvent, we can selectively study just the protein part, or the dodecylsulfate part, of the protein-dodecylsulfate complex. More than a thousandfold excess of dodecylsulfate (on a molar basis) is needed in order to dissociate alpha 2-macroglobulin to particles with, on average, half the original molecular mass. By combining the SANS data with results obtained by the equilibrium dialysis technique it follows that, under these circumstances, approximately one thousand dodecylsulfate molecules are associated per alpha 2-macroglobulin molecule. From the significant increase in the radius of gyration, which accompanies the dissociation process, we can conclude that the dissociation is associated with a drastic change in conformation of the protein molecule. From measurements where the dodecylsulfate part of the complex dominates the SANS signal we also get an indication that the dodecylsulfate is randomly distributed along the polypeptide chain, rather than being arranged in large clusters at certain regions of the protein molecule. By fitting the parameters of a binding model to the experimental data we obtain the result that most of the more than one thousand bound dodecylsulfate molecules, necessary for dissociation, are involved in the change in conformation, and the dissociation process is, in fact, driven by the binding of a very few extra dodecylsulfate molecules to the dissociation products. These data indicate that the dodecylsulfate-induced dissociation of alpha 2-macroglobulin is probably more complicated than just breaking, for instance, a hydrophobic interaction.