Trypsin Binding Activity Research Articles

Significance of D- tryptophan in Contryphan-Ar1131 Conus peptide: Oxidative folding, trypsin binding, and photostabilization activity

Distinct differences have been observed between L-tryptophan and D-tryptophan containing contryphan-Ar1131 in oxidative folding, trypsin binding, and photostabilization activity on avobenzone. [W5] contryphan-Ar1131 and [w5] contryphan-Ar1131 were chemically synthesized and characterized using RP-HPLC and mass spectrometry. Structural differences due to the change of configuration of tryptophan were evident from the optimized structures of contryphan-Ar1131 using density functional theory (DFT). The comparison of early events of oxidative folding has revealed the role of D-tryptophan in accelerating the formation of a disulfide bond. The optimized structures of the reduced form of peptides revealed the occurrence of aromatic-aromatic and aromatic-proline interactions in [w5] contryphan-Ar1131 which may be critical in aiding the oxidative folding reaction. The presence of the Lys6-Pro7 peptide bond indicates that contryphan-Ar1131 is resistant but may bind to trypsin allowing to assign the binding affinity of peptides to the protein surface. Competitive binding studies and molecular docking along with molecular dynamic (MD) simulations have revealed that [w5] contryphan-Ar1131 has more affinity for the active site of trypsin. Given tryptophan is a photostabilizer of FDA-approved chemical UV-A filter avobenzone, the report has compared the photostabilization activity of [W5]/ [w5] contryphan-Ar1131 on avobenzone under natural sunlight. [w5] contryphan-Ar1131 has better photostabilization activity than that of [W5] contryphan-Ar1131 and also individual D-tryptophan and L-tryptophan amino acids. These biochemical studies have highlighted the significance of D-tryptophan in contryphan-Ar1131 and its photostabilization activity on avobenzone may find applications in cosmetics.

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.

Differential isoform profiles of α2-macroglobulin from plasma of patients with chronic-progressive or relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is a human neurological disease for which no clinically useful marker has been identified in blood. This study examined α 2-macroglobulin ( α 2M) from the plasma of six patients with chronic-progressive MS and six with relapsing-remitting disease. The α 2M trypsin-binding activity in the plasma from both groups of patients did not differ from normal controls. However, after column isoelectric focusing, consistently less α 2M activity was recovered from the MS samples: those from the chronic-progressive and relapsing-remitting disease groups were an average of 43% and 68%, respectively, of controls. The number and isoelectric point (p I) values of the isoforms of the α 2M from patients with chronicprogressive disease were similar to controls. The average p I of the major form for both groups was 6.6. By contrast, the average p I of the major form from the patients with relapsing-remitting MS was significantly elevated to 7.1, and this group displayed a significantly higher percentage of total recovered activity above pH 7.0. In eleven of the twelve cases examined, the p I of the major form of α 2M correctly correlated with the clinical status of the patient. The original clinical diagnosis of the patients was reassessed by a 9-year retrospective interview which verified that 9 of the 10 patients in the follow-up group retained their original clinical diagnosis. These studies demonstrate differential isoform profiles of native α 2M from MS patients with progressive versus remitting disease which may be useful in subclassifying MS patients.

Kinetics of the urea-induced dissociation of human plasma α2-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 α2‐macroglobulin

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.

Biochemical Study of Protease Inhibitors in Normal Chinchilla Middle Ear

Protein concentration and inhibitory capacity of both alpha 1-antitrypsin (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M) were measured in plasma and middle ear bulla (MEB) washings of chinchillas by use of specific antisera against chinchilla alpha 1-AT and alpha 2-M. Low molecular weight (LMW) trypsin inhibitor also was analyzed in MEB washings. Chinchilla alpha 2-M showed a common antigenicity with human alpha 2-M. The mean value of alpha 1-AT in chinchilla plasma was 412.0 +/- 87.8 and that of alpha 2-M was 435.0 +/- 117.1 mg/dL. There was a significant relationship between alpha 1-AT level and antitryptic activity, and between alpha 2-M level and trypsin binding activity in plasma. The majority of alpha 1-AT and alpha 2-M in plasma is present as free inhibitors unsaturated with proteases. The MEB washings had significant antitryptic activity, which is attributed to both alpha 1-AT and LMW trypsin inhibitors. Inhibitory functions of alpha 1-AT and LMW trypsin inhibitors appear to play an important role in the defense of the normal middle ear mucosa.

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled α 2-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α 2-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat α 1-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-α 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.

Protease Inhibitors in Middle Ear Effusions from Experimental Otitis Media with Effusion: Kinetics of α1-Antitrypsin and α2-Macroglobulin Levels

Alpha 1-antitrypsin (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M) were measured by both immunochemical and functional assays in paired plasma and middle ear effusions (MEEs) from experimental otitis media models (serous otitis media [SOM], purulent otitis media [POM], and SOM + POM). The MEE/plasma ratio of alpha 1-AT in SOM was significantly higher than that in POM (p less than .01) because of the high alpha 1-AT level in POM plasma. The ratio of both antitryptic activity and trypsin-binding activity in POM was significantly higher than that in SOM + POM (p less than .01, less than .05), and significantly lower than that in SOM (p less than .01). The majority of both inhibitors in SOM exists as the free state, and the reaction between proteases and inhibitors in POM and SOM + POM is more active than that in SOM. It is concluded that both alpha 1-AT and alpha 2-M appear to play an important role in the protection of middle ear mucosa by forming protease-inhibitor complexes to reduce proteolytic damage in POM and SOM + POM models.

Protective effect of glucan-enhanced macrophage function in experimental pancreatitis

In an effort to assess the impact of enhanced macrophage function in acute pancreatitis, mice were subjected to a choline-deficient diet supplemented by ethionine to induce necrotizing pancreatitis. Treatment with the macrophage stimulant glucan resulted in improved survival rates (58 percent versus 14 percent) and maintenance of pancreatic architecture. Glucan treatment also resulted in decreased plasma and peritoneal trypsin activity, as well as increased trypsin-binding activity in the blood and peritoneal cavity. Plasma interleukin-1, as well as macrophage production of interleukin-1, were increased in the glucan-treated mice, which indicated enhanced macrophage function. These composite findings suggest that by enhancing diverse aspects of reticuloendothelial function, clinical use of immunomodulators may have significant impact on the pathogenesis of acute pancreatitis.

Altered isoelectric focusing of α 2-macroglobulin from plasma of patients with diabetes mellitus

Plasma from both type I and type II patients with diabetes mellitus displayed elevated α 2-macroglobulin trypsin-binding activity relative to normals. Column isoelectric focusing indicated that the average isoelectric point (pI) of the major form of crude α 2-macroglobulin was 6.6, 5.9, and 6.2 for normal, type I and type II subjects, respectively. Focused crude diabetic α 2-macroglobulin displayed significantly less recovered trypsin-binding activity relative to controls, particularly above a pI value of 6.0. Following preincubation with trypsin, the crude diabetic α 2-macroglobulin displayed a single isoelectric form (pI value of 5.3–5.5) and a substantial increase in recovered activity which were both comparable to normal α 2-macroglobulin. Purified α 2-macroglobulin from both normal and diabetic subjects displayed similar focusing profiles having a distribution of three forms with a pI value of 5.3–5.4 for the principal form. Trypsin-preincubated samples of purified α 2-macroglobulin displayed a single form with a pI value of 5.5–6.0. Preincubation of purified normal or diabetic α 2-macroglobulin with a plasma preparation devoid of α 2-macroglobulin from normal or diabetic plasma resulted in significantly lowered recovery of activity. All of the above studies suggest that the altered focusing properties observed for crude diabetic α 2-macroglobulin are due to components of the plasma rather than to α 2-macroglobulin itself.

Kinetics of the conformational alterations associated with nucleophilic modification of alpha 2-macroglobulin.

The effect of nucleophilic modification of alpha 2-macroglobulin (alpha 2M) with methylamine on the kinetics of sulfhydryl exposure was investigated. The generated sulfhydryl groups were detected with 4,4'-dithiodipyridine. The bimolecular rate constant for sulfhydryl exposure was determined to be 11.6 +/- 0.8 M-1 s-1 at 30 degrees C and pH 8.0. Treatment of alpha 2-macroglobulin with methylamine or proteases, such as plasmin and trypsin, results in a substantial increase in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid. This probe was used to monitor the kinetics of the conformational change occurring in alpha 2-macroglobulin upon treatment with methylamine. It was found that the conformational change did not occur simultaneously with the cleavage of the thiol ester bonds by the nucleophile but, rather, the conformational alterations occurred following a lag phase. The data are consistent with a mechanism requiring the random cleavage of two thiol ester bonds of a dimeric unit in the molecule prior to the unimolecular process representing the conformational change. According to this model, the two dimeric units present in alpha 2M act as independent entities. On the basis of the best fit with the model, the bimolecular rate constant, for hydrolysis of the thiol ester bonds, was determined to be 11.9 +/- 0.7 M-1 s-1, while the rate constant for the conformational change was (9.7 +/- 2.0) X 10(-3) s-1. The measured rate of conformational change is rate limited by thiol ester cleavage at lower methylamine concentrations. The conformational change, measured with this fluorescence probe, approximately parallels the loss of trypsin binding activity of alpha 2-macroglobulin, measured by resistance of the bound trypsin to soybean trypsin inhibitor. A much slower loss of plasmin binding activity was observed than was found for trypsin, suggesting that the structural requirements on alpha 2M for the interaction with plasmin are disrupted much more slowly than the structural requirements for trypsin binding upon methylamine treatment of the molecule.

Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes.

Human alpha 2-macroglobulin (alpha 2M) undergoes a conformational change after reaction with proteases. In this report, it is shown that although two trypsin molecules may bind simultaneously to each alpha 2M, only one trypsin is necessary to induce alpha 2M conformational change. Ternary complexes of alpha 2M and either two radioiodinated trypsins or two nonradioiodinated trypsins were purified by gel filtration chromatography. The nonradioactive complex did not bind 125I-trypsin, even after incubation for 24 h with the free protease present at a large molar excess. Under comparable conditions, a large molar excess of nonradioactive trypsin did not cause significant dissociation of the complex prepared with radioiodinated protease. Equations are presented that distinguish between two separate models of protease binding and demonstrate that binary alpha 2M-trypsin complex retains no significant trypsin binding activity despite the presence of a vacant protease binding site. Purified alpha 2M-plasmin complex, with 1.10 mol of plasmin/mol of inhibitor, also retained no trypsin binding activity as assessed with radioiodinated protein binding experiments. These studies suggest that reactions of alpha 2M with proteases are accurately described by the "trap hypothesis" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724) independent of protease size or binding stoichiometry.

Physical properties of human α2-macroglobulin following reaction with methylamine and trypsin

Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare α 2-macroglobulin, α 2-macroglobulin-trypsin complex and α 2-macroglobulin-methylamine complex. The circular dichroic spectrum of native α 2-macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of α 2-macroglobulin-trypsin and α 2-macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between α 2-macroglobulin-methylamine and native α 2-macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in α 2-macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with α 2-macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (s o 20,w) of 20.5 was determined for α 2-macroglobulin-methylamine compared to a value of 18.5 for unreacted α 2-macrogiobulin. This increase in sedimentation velocity is attributed to a 10% decrease in α 2-macroglobulin Stokes radius, α 2-Macroglobulin-trypsin complex prepared by reaction of the protease ata 2-fold molar excess with the inhibitor has a s o 20,w of 203. Although this sedimentation coefficient does reflect compacting of the α 2-macroglobulin structure compared to native α 2-macroglobulin, it is not large enough to rule out significant protrusion of the proteases from within pockets in the α 2-macroglobulin structure.

Functional modifications of alpha 2-macroglobulin by primary amines. Kinetics of inactivation of alpha 2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin.

The unique steric inhibition of endopeptidases by human alpha(2)M (alpha(2)-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by alpha(2)M was closely correlated with the loss of the methylamine-reactive sites in alpha(2)M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/alpha(2)M ratios, no unaccounted loss of trypsin-binding capacity occurred. As alpha(2)M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form alpha(2)M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [(14)C]methylamine with alpha(2)M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of alpha(2)M with time of methylamine treatment. It was found that conformational change of alpha(2)M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of (125)I-labelled trypsin to alpha(2)M did follow the same kinetics as the conformational change. This discrepancy between total binding ((125)I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound (125)I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of alpha(2)M and to the mechanism of formation of the complex.

Trypsin binding activity of alpha 2-macroglobulin in cystic fibrosis and other lung diseases.

Abnormalities of the interaction between alpha 2-macroglobulin and proteases have been suspected in cystic fibrosis. We measured the binding activity for trypsin of alpha 2-macroglobulin in 65 patients with cystic fibrosis, 41 obligate heterozygotes for cystic fibrosis, 18 children with asthma, 21 adult patients with chronic obstructive pulmonary disease, and 21 healthy control subjects. The assay was based on the observation that, once it is bound to alpha 2-macroglobulin, trypsin is no longer inhibited by soybean trypsin inhibitor. The bound trypsin retains activity against synthetic substrates such as N-alpha-benzoyl-arginine-p-nitroanilide. We found a moderately increased molar binding ratio of trypsin to alpha 2-macroglobulin in all patient groups and heterozygotes compared to healthy control subjects. The absolute concentration of alpha 1-macroglobulin was related to age rather than to the disease of the patient. Our data argue against a defect in protease-binding activity of alpha 2-macroglobulin in cystic fibrosis. The moderately increased binding activity observed could have been related to a structural difference in alpha 2-macroglobulin or to binding of ligands. This finding however, was not unique to patients with cystic fibrosis; the binding activity was increased in heterozygotes and in patients with other pulmonary diseases.

Physical and chemical properties of human plasma alpha2-macroglobulin.

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.

Hyper-alpha-2-macroglobulinemia in narcotic addicts.

Significant increases of serum alpha-2-macroglobulin (alpha-2-M) were detected in narcotic addicts presenting at a methadone treatment center. The mean alpha-2-M level was 341 +/- 14 mg/dl compared with 231 +/- 8 mg/dl in normal persons (p less than 0.01). In a comparable group of alcoholics with laboratory evidence of hepatic dysfunction the mean alpha-2-M level was 208 +/- 10 mg/dl. Although the serum immunoglobulin M content was also substantially elevated in the narcotic-addict group, no correlation was obtained between the serum content of this protein and alpha-2-M. Similarly, no correlation between alpha-2-M level and serum zinc content was observed. When the values of the trypsin-binding activity of serum measured in 13 addicts, 15 alcoholics with laboratory evidence of hepatic dysfunction, and 16 normal subjects were plotted against the amount of alpha-2-M measured in the same subjects, a linear correlation was obtained between trypsin-binding activity and alpha-2-M. Thus, the significantly increased serum trypsin-binding activity observed in the addicts is that which might be expected if normal alpha-2-M is being accumulated in large amounts as a result of increased macroglobulin synthesis.

Binding of zinc to alpha-2-macroglobulin and its role in enzyme binding activity

Physicochemical studies performed on alpha-2-macroglobulin were correlated with the biological activities of this protein. Equilibrium dialysis of the binding of 65Zn by alpha-2-macroglobulin at pH 7.9 showed heterogeneous binding which could be attributed to two classes of binding sites. The site of greatest affinity for zinc had an apparent stoichiometry ( n 1 in gatoms/mol of alpha-2-macroglobulin monomer) of 12 and an apparent association constant ( K 1) of 3.06·10 7. The second binding site had an n 2 of 60 and K 2 of 1.32·10 5. The trypsin binding activity of alpha-2-macroglobulin did not depend on the presence of zinc in this protein since all but traces of this metal could be removed by EDTA without loss of trypsin binding activity. Saturation of site 1 with zinc did not affect the trypsin binding activity of alpha-2-macroglobulin, but binding of the metal by site 2 progressively decreased the trypsin binding activity by causing an irriversable association of the alpha-2-macroglobulin molecules. Removal of excess zinc from alpha-2-macroglobulin did not restore its trypsin binding activity. Our results also indicate that the high zinc content of alpha-2-macroglobulin (320–770 μg/g protein) reported in the literature is an artifact and that native alpha-2-macroglobulin contains approximately 150–180 μg Zn/g protein.

The role of an α 2-macroglobulin of rat serum in the phagocytosis of colloidal particles

In these studies an α 1-an α 2-macroglobulin were isolated from rat serum. The α 2 protein had all the opsonic activity, promoting phagocytosis of various colloidal particles by rat liver slices. The opsonin was stable in the lyophilized form or in solution kept in ice. The opsonic activity was lost slowly at 37 °C at all pH values between 3.5 and 9.5. The activity increased when the protein was preincubated at 37 °C for 20 min. Measurement of optical rotatory dispersion suggested that no major change of conformation occurred when the temperature was raised to 37 °C. The protein dissociated, however, into two subunits at 37 °C. The activity was lost when the protein was incubated at 60 °C. The opsonin contained 6.5% carbohydrate comprised of hexosamine, hexose and sialic acid. Neuraminidase treatment released all the sialic acid without affecting the opsonic activity. Trypsin treatment inactivated the protein after prolonged incubation. The α 2-opsonic promoted phagocytosis of the test lipid emulsion of the reticuloendothelial system, colloidal gold and sulfur. It also stimulated the uptake of membrane fragments from heart, liver, skeletal muscle, skin and cancer cells. It is suggested that one of the most important physiological roles of the opsonin is to promote clearance of tissue debris produced under various adverse conditions. The uptake of two species of bacteria was also enhanced by the protein. The opsonin did not bind trypsin, all the trypsin binding activity was associated with the α 1-macroglobulin fraction. In this respect the α 2-opsonin is not related to the α 2-macroglobulin of human serum or to the α 2 acute phase macroglobulin of rat serum, both of which are known to bind proteolytic enzymes. Furthermore, an antiserum prepared against human α 2-macroglobulin did not show a precipitin line when tested against the opsonin even though it reacted weakly with rat serum indicating the presence of a cross-reacting antigen in rat serum. These data showed that the human α 2-macroglobulin and the rat α 2-opsonin are not related antigenically. The uptake of colloids in vitro required heparin as an obligatory cofactor, 5 units of heparin were optimal. Polyethylene sulfonate could replace heparin but chondroitin sulfate A and C were without effects.

Large scale purification of α2-macroglobulin from human plasma

A new convenient method for large scale preparation of pure α 2-macroglobulin from human plasma was described. The procedure involved precipitations with polyethylene glycol (300) and Rivanol followed by gel filtration. Ninety percent or more of the α 2-macroglobulin peak area, when concentrated to about 10 mg/ml, gave a single band in acrylamide gel electrophoresis, immunoelectrophoresis, and double immunodiffusion. The high degree of purity of our preparation was reflected also by the excellent correlation of results obtained from the determination of the protein content of the product by four different techniques (Lowry method, trypsin binding activity, radial immunodiffusion analysis, and absorbance measurement). The pure α 2-macroglobulin thus obtained was stable for months without change of trypsin binding activity.