Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy.

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.     

Steroid-protein interactions. XXXIV. Chemical modification of alpha1-acid glycoprotein for characterization of the progesterone binding site.

The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.     

Inflammation-induced expression of sialyl Lewisx is not restricted to α1-acid glycoprotein but also occurs to a lesser extent on α1-antichymotrypsin and haptoglobin

Acute and chronic inflammation-induced expression of sialyl Lewisx has already been shown to occur on α1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to α1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on α-antichymotrypsin and on haptoglobin. The level of expression of sialyl Lewisx on these proteins was lower than on α1-acid glycoprotein, in all likelihood because α1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl Lewisx was detectable on α1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated Lewisx was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the α3-linked fucose residues are present as sialyl Lewisx on α1-acid glycoprotein, α1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for α1-acid glycoprotein. This leads us to propose that α1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.     

Characterization of a sialyl alpha 2-3 transferase and a sialyl alpha 2-6 transferase from human platelets occurring in the sialylation of the N-glycosylproteins

Two sialyltransferases (EC 2.4.99.-) are extracted with Triton X-100 from human platelets and characterized with asialo 3H-labelled alpha 1-acid glycoprotein, an N-glycosylprotein. Methylation analysis of their specificities indicates that the enzymes transfer selectively sialic acid in a 3 or 6 position to oligosaccharides possessing Gal(beta 1-4)GlcNAc structure. The sialyl alpha 2-3 transferase was separated from the sialyl alpha 2-6 transferase by Ultrogel AcA34 column chromatography. Through affinity chromatography on CDPethanolamine-Sepharose, the two sialyltransferases are partly purified (5- and 20-fold enrichment of their specific activity, respectively, for sialyl alpha 2-3 transferase and alpha 2-6 transferase) and appear to be structurally heterogeneous.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

Specificity of sialytransferase: structure of alpha1-acid glycoprotein sialylated in vitro. A new methodology for the determination of the structure of the linkage formed by glycosyltransferase action on galactosyl-oligosaccharide-protein acceptors.

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.     

Controlled self-assembly of collagen fibrils by an automated dialysis system

In vitro self-assembled collagen fibrils form a variety of different structures during dialysis. The self-assembly is dependent on several parameters, such as concentrations of collagen and alpha1-acid glycoprotein, temperature, dialysis time, and the acid concentration. For a detailed understanding of the assembly pathway and structural features like banding pattern or mechanical properties it is necessary to study single collagen fibrils. In this work we present a fully automated system to control the permeation of molecules through a membrane like a dialysis tubing. This allows us to ramp arbitrary diffusion rate profiles during the self-assembly process of macromolecules, such as collagen. The system combines a molecular sieving method with a computer assisted control system for measuring process variables. With the regulation of the diffusion rate it is possible to control and manipulate the collagen self-assembly process during the whole process time. Its performance is demonstrated by the preparation of various collagen type I fibrils and native collagen type II fibrils. The combination with the atomic force microscope (AFM) allows a high resolution characterization of the self-assembled fibrils. In principle, the represented system can be also applied for the production of other biomolecules, where a dialysis enhanced self-assembly process is used.     

Sequence comparison of human plasma alpha1-acid glycoprotein and the immunoglobulins

The amino acid sequence of human plasma alpha1-acid glycoprotein, upon comparison with the sequences of other blood proteins, was shown to possess significant similarity with the immunoglobulins. Employing direct and corrected sequence identity, the average mutation value and two different computer comparisons for the evaluation of sequence similarity, the following two regions of this alpha-globulin, which account for approximately half of the total amino acid sequence of the protein, were found to possess sequence similarity with the immunoglobulins. a) The region from residues 77 through 125 proved to be related to the variable region of several human H and L chains, and b) the region from residues 136 through 166 was found to be related not only to the constant region of a human and a mouse L chain but also to the third and fourth constant region of a rabbit and a human H chain, respectively. These results suggest that alpha1-acid glycoprotein is probably related to the immunoglobulins and further suggest that it possibly diverged from the immunoglobulin evolutionary tree prior to the formation of the primitive L chain.     

Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy

The ultrastructure of fibrous long spacing (FLS) collagen fibrils has been investigated by performing both atomic force microscopy (AFM) and transmission electron microscopy (TEM) on exactly the same area of FLS collagen fibril samples. These FLS collagen fibrils were formed in vitro from type I collagen and alpha1-acid glycoprotein (AAG) solutions. On the basis of the correlated AFM and TEM images obtained before and after negative staining, the periodic dark bands observed in TEM images along the longitudinal axis of the FLS collagen fibril correspond directly to periodic protrusions seen by AFM. This observation is in agreement with the original surmise made by Gross, Highberger, and Schmitt (Gross J, Highberger JH, Schmitt FO, Proc Natl Acad Sci USA 1954;40:679-688) that the major repeating dark bands of FLS collagen fibrils observed under TEM are thick relative to the interband region. Although these results do not refute the idea of negative stain penetration into gap regions proposed by Hodge and Petruska (Petruska JA, Hodge AJ. Aspects of protein structure. Ramachandran GN, editor. New York: Academic Press; 1963. p. 289-300), there is no need to invoke the presence of gap regions to explain the periodic dark bands observed in TEM images of FLS collagen fibrils.     

Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma alpha(1)-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1-->4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2-->2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl-->N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas-liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.     

Collagen polymorphism: its origins in the amino acid sequence.

The polymorphic forms of ordered collagen aggregation in vitro and in vivo are reviewed. The axially projected structures of a class of fibrils known as fibrous long spacing (FLS) collagen are solved using simulated positively stained banding patterns based on the amino acid sequence. This method is also used to solve the axial projection of a 670 Å (D) periodic structure with a symmetrical banding pattern (DPS) re-precipitated from skin collagen. The relation between the obliquely striated and 110 Å periodic forms of collagen is discussed. The specificity for the formation of FLS, DPS and segment long spacing (SLS) collagen is shown to be in the distributions of various amino acids in the sequence. Different residues are important for each type of structure, their importance being dependent on the chemical conditions and the presence of other macromolecules. The interaction of collagen fibrils with proteoglycans in vivo is discussed in terms of the amino acid sequence. Also the factors which affect collagen morphology in the presence of mucopolysaccharides and proteoglycans in vitro and in vivo are discussed. Some insight is gamed into the principles which govern the self-assembly of molecules into ordered fibrous aggregates.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.     

Topography of human plasma alpha1-acid glycoprotein.

Human plasma alpha1-acid glycoprotein, whose linear amino acid sequence has recently been elucidated (Schmid et al. (1973), Biochemistry 12, 2711), was further investigated with regard to its topography. Nitration of this protein and subsequent elucidation of the structures of the peptides containing modified tyrosine indicated that residues 27, 37, 78, 115, 127, and 157 are free, 50 and 91 are in an intermediate state, and 65, 74, 110, and 142 are buried. CD measurements between pH 10 and 12 demonstrated that the buried tyrosines are strongly hydrogen bonded and are probably responsible to a considerable extent for the stability of this protein. Of the three tryptophans of this protein, residue 122 proved to be partially reactive with Koshland reagent while the other two (25 and 160) were found to be unreactive. The state of the two disulfide bonds, established by differential reduction and alkylation with specific reagents, was shown to be of an intermediate type. Using carboxymethylation with bromoacetate at pH 7.0 for 8 days, the three histidines (97, 100, and 171) and methionine 111 could be shown to be in intermediate states. All lysines were treated with trinitrobenzenesulfonate and thus were assumed to be free. Of the 40 carboxylic groups, which were amidated with glycine methyl ester, 32 including the 14 sialyl residues were found to be free, six in an intermediate and the remaining two in a buried state. The present study describes the states of almost half of the amino acid residues of alpha1-acid glycoprotein, a knowledge important for the construction of a preliminary three-dimensional model of this conjugated protein.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

Ultrastructural aspects of corneal fibrous tissue in the scheie syndrome

An ultrastructural study of the corneal fibrous tissue was performed in a case of Scheie's syndrome. Mucopolysaccharidosis deposits in keratocytes were observed as electron-clear and electron-dense inclusions. Modifications of the extracellular space included modifications of lamellar collagen organization and local hypertrophy of collagen bundles; presence of microfibrillar dense material isolating large irregular collagen fibers; and presence of fibrous long spacing type collagen fibers. The significance of these changes is discussed. This special form of collagen organization is supposed to appear in a modified microenvironment, that is the presence of an abnormal concentration of proteoglycans.     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.