Amino acid sequence homology between bovine and human platelet proteins

The complete amino acid sequence of bovine platelet factor 4 (PF-4) was determined. Comparison of the 88 residue bovine protein with its 70 residue human counterpart indicated 73% homology. There is 53% homology between this bovine protein and another human platelet protein, beta-thromboglobulin (beta-TG). These heparin binding proteins share greatest homology around a lysine-rich octa-peptide near the carboxy-terminus which is the putative heparin binding domain. Graphic comparison of these proteins suggests that a point mutation at position 55 (human PF-4 numbering) could cause a significant difference among the folding properties of these 3 proteins and might be critical for their different heparin binding properties.     

Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.     

Conversion of low-affinity platelet factor 4 to beta-thromboglobulin by plasmin and trypsin

Low-affinity platelet factor 4 and beta-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that beta-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as beta-thromboglobulin. We conclude that beta-thromboglobulin is a breakdown product of low-affinity platelet factor 4.     

Complete covalent structure of human beta-thromboglobulin.

The complete primary structure of the platelet-specific protein human beta-thromboglobulin has been determined. beta-Thromboglobulin consists of identical subunits of 81 amino acids, each with a molecular weight of 8851. The amino acid sequence of the beta-thromboglobulin subunit is: Gly-Lys-Glu-Glu-Ser-Leu-Asp-Ser-Asp-Leu-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr-Thr-Ser-Gly-Ile-His-Pro-Lys-Asn-Ile-Gln-Ser-Leu-Glu-Val-Ile-Gly-Lys-Gly-Thr-His-Cys-Asn-Gln-Val-Glu-Val-Ile-Ala-Thr-Leu-Lys-Asp-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Pro-Asp-Ala-Pro-Arg-Ile-Lys-Lys-Ile-Val-Gln-Lys-Lys-Leu-Ala-Gly-Asp-Glu-Ser-Ala-Asp. Disulfide bridge-18 to half-cystine-58. The amino acid sequence of beta-thromboglobulin shows a marked homology with that of platelet factor 4. When the sequences are aligned for maximum homology, 42 of the 81 residues of beta-thromboglobulin are identical with those of platelet factor 4, including the position of the four half-cystines.     

Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and beta-thromboglobulin

Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/beta-thromboglobulin (LA-PF4/beta TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling beta TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of beta TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total beta TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. beta TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.     

Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.     

Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin

Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu-Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.     

Identification and characterization of a phospholipid-binding site of bovine factor Va

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.     

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M_r calculated from the deduced as sequence was 113 383. Bovine F3 had 93, 94 and 77% as identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two as residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.     

Interspecies conservation of structure of interphotoreceptor retinoid-binding protein. Similarities and differences as adjudged by peptide mapping and N-terminal sequencing.

Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human proteins, whereas both sets differ considerably from the bovine protein pattern. Time-course digestion shows monkey IRBP to be more readily cleaved than bovine IRBP and also cleaved to smaller fragments. Also, reversed-phase h.p.l.c. of complete tryptic digests of the IRBPs indicate that, although they have in common a similar preponderance of hydrophobic peptides, all three proteins differ extensively in their fine structure. The N-terminal sequences of monkey and bovine IRBPs have been extended beyond those presented in our previous report [Redmond, Wiggert, Robey, Nguyen, Lewis, Lee & Chader (1985) Biochemistry 24, 787-793] to over 30 residues each. The sequences yet show extensive homology, differing at only two positions, although the major monkey sequence has an additional five amino acid residues at its N-terminus ('n + 5' sequence) not observed with bovine IRBP ('n' sequence). The newly determined N-terminal sequence of human IRBP demonstrates the presence of equal amounts of the 'n' and 'n+5' sequences that are qualitatively identical with those of the monkey. The presence of the five-amino-acid-residue extension in primate, but not bovine, IRBP may indicate variation in post-translational processing.     

Amino acid sequence of the carboxy-terminal end of human erythrocyte glucose-6-phosphate dehydrogenase

Human erythrocyte glucose-6-phosphate dehydrogenase was purified to homogeneity by a simplified procedure, consisting of 2',5'-ADP-Sepharose affinity chromatography, followed by Sephadex G-100 gel filtration. The carboxy-terminal region of the protein was identified by carboxypeptidase digestion: the sequence -Lys-Leu-COOH was found instead of the reported -Gly-COOH, thus showing identity with the carboxy-terminal sequence of glucose-6-phosphate dehydrogenase from human leukocytes and platelets. In addition, the carboxyl-terminal peptide was isolated from a tryptic digest of the protein and sequenced. The sequence is: Trp-Val-Asp-Pro-His-Lys-Leu.     

Purification and characterization of human and bovine platelet factor 4.

Platelet antiheparin, platelet factor 4, was isolated from freeze-thaw lysates of fresh bovine and outdated human platelet concentrates by a single step affinity chromatographic procedure. The yields of PF4 were 93 microgram and 142 microgram/ml of human and bovine platelets respectively. Antiheparin activity of the products were 558 units/mg for the bovine isolate and 489 units/mg for the human material. The bovine product is a single chain polypeptide with an apparent molecular weight of 12,300. Amino acid composition indicates 107-109 residues compared to the smaller human product which has an apparent molecular weight of 8,000 for a 70 residue polypeptide. The intact polypeptide was resistant to enzymatic hydrolysis as opposed to the reduced-alkylated derivative which was susceptible to hydrolysis in the presence and absence of heparin.     

Isolation, characterization, and immunological detection of neutrophil-activating peptide 2: a proteolytic degradation product of platelet basic protein.

Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25-94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or beta-thromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1-1.5 microns) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, beta-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77-94) were inactive, whereas platelet basic protein fragment 22-89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.     

Complete primary structure of prostatropin, a prostate epithelial cell growth factor

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.     

Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1.

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.