Antigenic determinants of bovine myelin encephalitogenic protein recognized by rabbit antibody to myelin encephalitogenic protein.

The antigenic determinants of bovine myelin encephalitogenic protein were investigated by quantitative complement fixation and hapten inhibition using rabbit anti-monkey protein and anti-bovine protein and purified and characterized fragments of bovine protein. The two regions of bovine encephalitogenic protein containing determinants were sequences 1 to 43 and 90 to 170. One rabbit antiserum recognized a determinant(s) probably in residues 15 to 40 while for another rabbit antiserum the determinant of 1 to 43 resided in 1 to 20. The determinant(s) of residues 90 to 170 involved the region around the tryptophan at position 116. Fragment 44-89, which contains the major encephalitogenic determinant for the rabbit, was virtually devoid of any reactivity with the rabbit anti-encephalitogenic protein. It appears that portions of the protein other than the encephalitogenic site are responsible for stimulation of bone marrow-derived cells and antibody production. In demonstrating selected regions of the protein as sites for antigenic determinants, the present immunochemical studies also suggest that the protein might have a more folded conformational alignment than previously suspected.     

Proteolytic and peroxidatic reactions of commercial horseradish peroxidase with myelin basic protein

Degradation of myelin basic protein during incubations with high concentrations of horseradish peroxidase has been demonstrated [Johnson & Cammer (1977) J. Histochem. Cytochem.25, 329-336]. Possible mechanisms for the interaction of the basic protein with peroxidase were investigated in the present study. Because the peroxidase samples previously observed to degrade basic protein were mixtures of isoenzymes, commercial preparations of the separated isoenzymes were tested, and all three degraded basic protein, but to various extents. Three other basic proteins, P(2) protein from peripheral nerve myelin, lysozyme and cytochrome c, were not degraded by horseradish peroxidase under the same conditions. Inhibitor studies suggested a minor peroxidatic component in the reaction. Therefore the peroxidatic reaction with basic protein was studied by using low concentrations of peroxidase along with H(2)O(2). Horseradish peroxidase plus H(2)O(2) caused the destruction of basic protein, a reaction inhibited by cyanide, azide, ferrocyanide, tyrosine, di-iodotyrosine and catalase. Lactoperoxidase plus H(2)O(2) and myoglobin plus H(2)O(2) were also effective in destroying the myelin basic protein. Low concentrations of horseradish peroxidase plus H(2)O(2) were not active against other basic proteins, but did destroy casein and fibrinogen. Although high concentrations of peroxidase alone degraded basic protein to low-molecular-weight products, suggesting the operation of a proteolytic enzyme contaminant in the absence of H(2)O(2), incubations with catalytic concentrations of peroxidase in the presence of H(2)O(2) converted basic protein into products with high molecular weights. Our data suggest a mechanism for the latter, peroxidatic, reaction where polymers would form by linking the tyrosine side chains in basic-protein molecules. These data show that the myelin basic protein is unusually susceptible to peroxidatic reactions.     

The horseradish peroxidase technique for cell lineage studies.

The identification of cell lineage for a given cell type of a particular tissue is an important step in understanding how this process contributes to histogenesis. The importance in understanding cell lineage has relevance for both theoretical and practical reasons. For example, delineating and identifying histogenic principals is required to advance stem cell research and tissue regeneration. To document cell lineage in a given experimental preparation, a number of techniques have been developed. This typically requires the injection of a tracer compound into a founder cell. As this cell produces progeny, the tracer is passed on to the daughter cells. By detecting the tracer in the progeny or daughter cells the investigator can determine which cells originated from the cell that was originally injected with the tracer. By using such an approach it is possible to trace the developmental path from the first cells of the embryo to the specialized cells making the tissue of the adult. A number of tracer compounds have been used with good results in lineage tracing. One of these tracer compounds is horseradish peroxidase (HRP). Several variations of the technique are available depending on what species are studied or what histological requirements are made by the study. A basic technique that can be adapted to individual needs is presented. Included in this protocol on lineage tracing are the procedures for injection, fixation, and the microscope evaluation of labelled cells in the Helobdella triseralis embryo. A brief discussion of the technique will note its advantages and disadvantages. Examples of labelled cell preparations are given to illustrate the technique.     

The effects of glutaraldehyde treatment and horesradish peroxidase conjugation on the immunoreactivity of bovine myelin encephalitogenic protein.

The effects of the immunoreactivity of bovine myelin encephalitogenic protein (EP) of treatment with glutaraldehyde and conjugation with horeradish peroxidase (HRP) were investigated by complement fixation and immunohistochemistry. Both glutaraldehyde treatment and HRP conjugation of EP decreased but did not abolish the reactivity of EP with rabbit anti-EP. Conjugation of EP to HRP by the two-step method of Avrameas had less detrimental effects on the immunochemical reactivity of EP than did the one-step procecure. The use of EP-HRP conjugates to probe for antibodies to EP is an immunochemically sound system with the major limitation that the number of antibody-producing cells is likely to be underestimated due to the failure to detect cells producing low levels of antibody or antibodies directed toward determinants altered by the modification procedures.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

NMR studies of myelin basic protein. X. Conformation of a determinant encephalitogenic in the rabbit

Residues 67 to 75 in myelin basic protein from several species comprise the sequence Thr-His-Tyr-Gly-Ser-Leu-Pro-Gln-Lys that acts as an encephalitogenic determinant in the rabbit. Proton magnetic resonance spectra of human, bovine and porcine proteins display nuclear Overhauser effects between the delta-CH of Tyr-69 and the delta-CH3 of Leu-72, which indicate reverse-turn conformations about the Gly-Ser residues. This effect occurs also in physiological saline solution at pH 6.0 but in dimethylsulfoxide solution the nuclear Overhauser effect disappears. Circular dichroism indicates that the protein when bound to ganglioside micelles acquires 30-40% alpha-helical conformation, but the reverse turn still persists in the sequence of the rabbit encephalitogen. These results suggest that the encephalitogenic region of the protein remains at the aqueous interface of the micelles.     

EPR studies on compound I of horseradish peroxidase.

Compound I of horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase EC 1.11.1.7) was studied by EPR at low temperatures. An asymmetric signal was found, about 15 Gauss wide and with a g-value of 1.995, which could be detected only at temperatures below 20 K and which had an intensity corresponding to about 1% of the heme content. In a titration with H2O2, the signal intensity was proportional to the concentration of Compound I, reaching a maximum when equivalent amounts of H2O2 were added. This indicates that the signal is not due to an impurity, and it is suggested that a free radical is formed, relaxed by a near-by fast-relaxing iron.     

Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.     

The major site of guinea-pig myelin basic protein encephalitogenic in Lewis rats.

In the Lewis rat, fragment 43–88 of the highly encephalitogenic guinea-pig basic protein has been previously shown to retain the full activity of the parent protein. In the present studies this fragment was subjected to controlled chymotryptic digestion so that cleavage occurred only at tyrosine 67, generating two peptides, residues 43-67 and residues 68-88. When compared on an equimolar basis peptide 68-88 had the same encephalitogenic activity as the intact fragment and induced the same degree of immunologically specific cell response as measured by the in vitro lymphocyte stimulation test. Peptide 68-88 was further fragmented by selective tryptic cleavage at arginine 78 after blocking lysine 73 with citraconic anhydride. The two peptides, residues 68-78 and residues 79-88, were not encephalitogenic, indicating that residues adjacent to the point of cleavage contribute to the active site.     

Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol-aniline coupling hydrogen donor

The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.     

The ordered structure of the encephalitogenic protein from normal human myelin

The conformation of the encephalitogenic protein isolated from normal human myelin has been studied by circular dichroism and surface tension techniques. The findings support the conclusion that this protein has a highly ordered structure in solution, with little α-helical or β structure. Conformational changes were observed at extremes of pH. Heating at high or low pH values had the effect of inducing more structure as determined by circular dichroism. Surface tension measurements showed a low temperature conformational change at low pH and a high temperature conformational change at high pH. At other pH values the structure appeared to be stable.     

Amino acid sequence of the encephalitogenic basic protein from human myelin

Myelin from the central nervous system contains an unusual basic protein, which can induce experimental autoimmune encephalomyelitis. The basic protein from human brain was digested with trypsin and other enzymes and the sequence of the 170 amino acids was determined. The localization of the encephalitogenic determinants was described. Possible roles for the protein in the structure and function of myelin are discussed.