Minor Structural Alterations Research Articles

Silicone Oil Induced Aggregation of Proteins

Prior to delivery to the patient, protein pharmaceuticals often come in contact with a variety of surfaces (e.g., syringes and stoppers), which are treated to facilitate processing or to inhibit protein binding. One such coating, silicone oil, has previously been implicated in the induction of protein aggregation. We have investigated the propensity of model proteins to aggregate when silicone oil is present in solution and find significant induction of aggregation in four proteins of various molecular weights and isoelectric points in the presence of 0.5% oil. The ability of silicone oil to induce conformational changes that might be responsible for this aggregation was also examined by a combination of circular dichroism (CD) and derivative UV spectroscopy. Neither method produces evidence of large conformational changes or alterations in thermal stability although in a limited number of cases some small changes suggest the possibility of minor structural alterations. The most probable explanation for silicone oil induced aggregation is that the oil has direct effects on intermolecular interactions responsible for protein association through interaction with protein surfaces or indirectly through effects on the solvent.

Ultrasound-Enhanced Transcorneal Drug Delivery

Ultrasound has been shown to enhance, by up to 10 times, the corneal permeability to different compounds such as beta-blockers and fluorescein. Here, we report on our investigation of the mechanisms of ultrasound-enhanced drug delivery through the cornea using light and electron microscopy. Enhancement of permeability for a hydrophilic compound, sodium fluorescein, in rabbit cornea in vitro was achieved using ultrasound at a frequency of 880 kHz and intensities of 0.19-0.56 W/cm2 with an exposure duration of 5 minutes. Light and electron microscopy (transmission and scanning) were used to observe ultrasound-induced structural changes in the cornea. The permeability increased by 2.1, 2.5, and 4.2 times when ultrasound was applied at 0.19, 0.34, and 0.56 W/cm2, respectively (P<0.05). The surface cells of corneal epithelium exposed to ultrasound appeared swollen and lighter in color (indications of membrane rupture) as compared with the control cells. Some of the surface epithelial cells were absent. The cells in the inner layers of the epithelium were occasionally lighter in color. Also, holes 3-10 microm in diameter were observed on the epithelial surface. No structural changes were observed in the stroma. Ultrasound enhancement of drug delivery through the cornea appears to result from minor structural alterations in the epithelium. Careful investigation of the recovery of cornea structure and barrier function after the ultrasound application, in vivo, is needed.

The Differential Cytotoxicity of Water-Soluble Fullerenes

We show that the cytotoxicity of water-soluble fullerene species is a sensitive function of surface derivatization; in two different human cell lines, the lethal dose of fullerene changed over 7 orders of magnitude with relatively minor alterations in fullerene structure. In particular, an aggregated form of C 60, the least derivatized of the four materials, was substantially more toxic than highly soluble derivatives such as C3, Na+ 2-3[C60O7-9(OH)12-15] (2-3)- , and C60(OH)24. Oxidative damage to the cell membranes was observed in all cases where fullerene exposure led to cell death. We show that under ambient conditions in water fullerenes can generate superoxide anions and postulate that these oxygen radicals are responsible for membrane damage and subsequent cell death. This work demonstrates both a strategy for enhancing the toxicity of fullerenes for certain applications such as cancer therapeutics or bactericides, as well as a remediation for the possible unwarranted biological effects of pristine fullerenes.

O modo de coaptação glótica em crianças no diagnóstico diferencial de alteração estrutural mínima

A configuração glótica varia durante a fonação, mesmo em indivíduos sem queixas vocais e sem alterações ao exame, de acordo com a idade, o sexo, o registro vocal, a freqüência fundamental, a tensão e as lesões. Observa-se junção completa ou incompleta da borda livre da prega vocal; quando incompleta, formam-se fendas de formatos variados. OBJETIVO: Nosso objetivo é encontrar no modo de coaptação glótica, durante a fonação sustentada da vogal /épsilon/ em crianças com alteração estrutural mínima, elementos que permitam diferenciá-lo dos indivíduos com nódulo vocal ou de indivíduos sem queixas vocais. MATERIAL E MÉTODO: Estudo retrospectivo de dados de crianças atendidas no período de 1996 a 2001. A amostra constou de imagens de laringes de crianças que apresentaram diagnóstico de alteração estrutural mínima, nódulo vocal e também de crianças sem queixas vocais, estes denominados normais. Destas imagens foi analisada a configuração glótica durante a fonação da vogal /épsilon/ e realizada análise estatística para a comparação entre os três grupos. RESULTADOS: As fendas triangulares são encontradas nos três grupos, enquanto que a fenda fusiforme só ocorreu nas alterações estruturais mínimas. CONCLUSÕES: A utilização do modo de coaptação glótica em crianças, como critério diagnóstico para diferenciar alteração estrutural mínima de nódulo vocal e de laringe normal, é relevante quando se observa fenda fusiforme, condição encontrada somente nas alterações estruturais mínimas; as fendas triangulares não se mostraram significantes para diferenciar alteração estrutural mínima de nódulo vocal e laringe normal.

DNA Binding Mode of the Isoquinoline Alkaloid Berberine with the Deoxyoligonucleotide d(GCCGTCGTTTTACA)2

The ability of protoberberine alkaloids, berberine and berberrubine, to act as topoisomerase II poisons is linked to the anti-cancer activity. Minor alterations in structure have a significant effect on their relative activity. Berberine, which has methoxy group at the 19-position, is significantly less potent than berberrubine. Several observations support non-specific binding to HP14 by the berberine: (i) nonspecific upfield changes in <TEX>$^1H$</TEX> chemical shift for protons of the berberine; (ii) the broadening of imino protons of HP14 upon binding of the berberine; (iii) very small increases in duplex melting temperature in the presence of the berberine. Our results reveal that substitution of a hydroxyl group to a methoxy group on the 19-position, thereby converting the berberrubine to the berberine is associated with a non-specific DNA binding affinity and a reduced topoisomerase II poisoning. The presence of a bulky 19-methoxy substituent decreases intercalating properties of berberine and makes it inactive as topoisomerase II poison.

Cytogenetic Studies of Some F1 Hybrids between Wild and Cultivated Taxa of Capsicum L.

Two F1 hybrids one between wild C. annuum var glabrisculum and C. frutescens (H1) and another between wild C. annuum var antigua and C. frutescens (H2) were obtained. Cytogenetic analysis of F1 hybrids showed that the parental genomes differ from each other by 2 or 1 translocations, 1 inversion and some minor structural alterations leading to reduced homologies between the respective parental genomes. Meiotic irregularities pollen and seed sterilities were higher in H2 than in H1. Probable reasons for the differences in respect of their crossability relationships, chromosome pairing and fertilities in non-rearing of reciprocal hybrids are suggested. The role of chromosome structural changes in genome differentiation is suggested. Isolating mechanisms viz., hybrid inviability and hybrid weakness in the reciprocal cross and hybrid sterility in the F1 hybrids are in operation.

Single additional methylene group in the head-group region imparts high gene transfer efficacy to a transfection-incompetent cationic lipid

In combination with equimolar 1,2-dioleoyl- L-α-glycero-3-phosphatidyl ethanolamine, a novel cholesterol-based cationic lipid with β-alanine head-group ( 2) has been demonstrated to be strikingly more efficacious (10–24-fold) in transfecting CHO, COS-1 and HepG2 cells than its glycine analog ( 1) containing just one less methylene unit in its head-group region. Syntheses, characterizations and in vitro transfection biology of lipids 1 and 2 are described. Present findings demonstrate that even truly minor structural alterations, such as inclusion of just one additional methylene functionality in the polar head-group region, can convert an essentially transfection-incompetent cholesterol-based cationic amphiphile to a remarkably efficient cationic transfection lipid.

A criança disfônica: diagnóstico, tratamento e evolução clínica

Os nódulos vocais são causas freqüentes de disfonias infantis, porém, através das vídeoendoscopias, outras lesões têm sido diagnosticadas na laringe infantil, como os cistos, sulcos, pontes e micromembranas, denominadas de lesões estruturais mínimas. OBJETIVO: Avaliar, em 71 crianças com disfonia agendadas nos ambulatórios de Foniatria da Faculdade de Medicina de Botucatu (Unesp) nos últimos cinco anos: sexo, idade, diagnósticos, tratamentos e evolução clínica. FORMA DE ESTUDO: Clínico retrospectivo. CASUÍSTICA E MÉTODO: Realizou-se estudo retrospectivo das avaliações otorrinolaringológicas e endoscópicas de 71 crianças com queixas de disfonia e idade entre 3 a 13 anos (45 meninos e 26 meninas). As crianças haviam sido submetidas a exame endoscópico com telescópio rígido ou nasofibroscópio flexível. Foram excluídas as crianças com disfonias funcionais ou com queixas de obstrução respiratória. RESULTADOS: Os principais diagnósticos foram: nódulo vocal (47 casos; 66,2%), cisto epidérmico (7 casos; 9,9%), cisto aberto fistulizado (6 casos; 8,4%), sulco vocal (5 casos; 7,1%), ponte + cisto (2 casos; 2,8%), micromembrana anterior (2 casos; 2,8%) e pólipo vocal (2 casos; 2,8%). As disfonias foram mais freqüentes entre os meninos (63,3%). A fonoterapia isolada foi o tratamento de escolha nos casos de nódulo vocal (evolução favorável), sulco vocal (evolução desfavorável) e micro-membrana (evolução desfavorável). Nas demais lesões (cistos, pontes e pólipos) o tratamento de escolha foi a cirurgia associada à fonoterapia, com resultados satisfatórios, exceto nos casos que recusaram a cirurgia. CONCLUSÃO: Os nódulos vocais são as principais causas de disfonias em crianças, porém as lesões estruturais mínimas mostraram-se também freqüentes, exigindo exame minucioso para o diagnóstico.

Crystal structure and stability studies of C77S HiPIP: a serine ligated [4Fe-4S] cluster.

The crystal structure of Chromatium vinosum C77S HiPIP has been determined and is compared with that of wild type. This is the first reported crystal structure of a Ser ligated [4Fe-4S] cluster and reveals a 0.11 A shortening of the Fe-O bond (relative to Fe-S), but only minor structural alterations of the overall tertiary structure. Coordination changes are corroborated by resonance Raman spectroscopy. Comparison of the crystal and solution structures for HiPIPs identifies Phe48 as the main controller of solvent access to the Fe-S cluster; however, there is no significant change in cluster solvation of the C77S mutant relative to WT HiPIP. Ser ligation ultimately results in decreased cluster stability due to increased sensitivity to proton mediated degradation.

Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor

Metal‐induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc‐induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc‐induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal‐induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 1480–1485, 2000

A comparative study of fossil and extant algaenans using ruthenium tetroxide degradation

In this study the chemical structure of algaenans isolated from the freshwater algae Tetraedron minimum, Pediastrum boryanum and Botryococcus braunii are compared with their fossil counterparts by means of RuO 4 oxidation. The results show that the algaenans investigated are preserved in sediments with only minor structural alterations. However, product mixtures from RuO 4 degradation of the fossil algaenans exhibit a broader distribution of oxidation products than freshly isolated algaenans indicating that the fossil biopolymers contain a greater proportion of ether cross-links, which maybe an effect of diagenetic alteration or different algal strains. Despite these differences, fossil algaenans can still be recognised chemically on the basis of the specific RuO 4 oxidation products, even after 50 Ma of sediment burial.

Hypoxic Hypoperfusion Fails to Induce Myocardial Hibernation in Anesthetized Swine.

BACKGROUND: Congenital origin of the left coronary artery from the pulmonary artery (ALCAPA) results in chronically dysfunctional myocardium with the partial ability to recover after revascularization. We attempted to establish an ALCAPA syndrome in anesthetized pigs for 24 hours and to compare it with stunned and infarcted myocardium. METHODS AND RESULTS: In group 1 (n = 12), a bypass graft was interposed between the pulmonary artery and the left anterior descending coronary artery (LAD). Reduction of flow in the LAD with gradual increases in flow from the pulmonary artery resulted in an incremental reduction of segment shortening (8.9 +/- 5.3% at 24 hours vs 26.6 +/- 10% at baseline, P <.005). In group 3 (n = 5), 2 cycles of 10-minute LAD occlusion resulted in decreased segment shortening with slow recovery (at 24 hours 18.7 +/- 1.3% vs 24.2 +/- 4% at baseline, segment shortening with slow recovery (at 24 hours 18.7 +/- 1.3% vs 24.2 +/- 4% at baseline, P <.05). In group 3 (n = 6), 1-hour LAD occlusion reduced segment shortening at 24 hours to 4.7 +/- 5.2% (P <.005 vs baseline). Histological analysis of the LAD territory revealed severe degeneration, myolysis, and alteration of the chromatin structure in group 1 comparable to ischemic cell death in group 3, whereas control areas and the LAD area in group 2 showed only minor structural alterations. Infarct size/risk area, as measured by tetrazolium staining, was 49.8 +/- 11.2% in group 1, 9.3 +/- 8.1% in group 2 (P <.005), and 60.3 +/- 9% in group 3. CONCLUSION: Hypoxic myocardial hypoperfusion from the pulmonary artery results in myocardial necrosis in anesthetized pigs. These findings are in contrast to the concept of myocardial hibernation in the ALCAPA syndrome because in this model, hypoxic hypoperfusion failed to induce adaptation to preserve myocardial structure.

Fourier Analysis of Textural Variations in Human Normal and Cataractous Lens Nuclear Fiber Cell Cytoplasm

Variations in cytoplasmic texture of human normal and cataractous lenses were investigated with Fourier analysis. Fixed Vibratome sections of six normal transparent human lenses (age range 55–72 years) and six nuclear cataractous lenses (age range 66–89 years) were examined using transmission electron microscopy. Images were taken of the fiber cell cytoplasm and examined using linear optical density scans, Fourier transforms and autocorrelation analysis. The cytoplasm of normal human lenses was found to be smooth and homogeneous. Radially-averaged plots of Fourier transforms revealed a broad band of intensity over the range of 15–50 nm. Four of the six cataracts also had homogeneous cytoplasm without substantial density variations. Fourier transforms and radially-averaged plots were similar to those of the normal, although slightly lower in intensity for components⩽20 nm. Two of the six cataracts had a slightly granular or textured cytoplasm that showed significant density variations. Fourier transforms and radially-averaged plots revealed significant intensity in the range⩾200 nm. Autocorrelation analysis supported the data obtained by Fourier transforms. Radially-averaged plots of the autocorrelation resultant displays detected only small structural units in the normal and non-textured cataractous cytoplasm, whereas larger structural units were detected in the textured cataractous cytoplasm. The appearance of the textured cataractous cytoplasm suggests that redistribution or loss of protein may be the cause of density variations in these types of cataracts. Importantly, these results indicate that some nuclear cataracts do not contain spatial fluctuations in the cytoplasm large enough to cause significant light scattering. The data suggest that nuclear cataractogenesis is a multi-factorial process involving minor alterations in cellular structure.

Structure-function analysis of a conserved aromatic cluster in the N-terminal domain of human epidermal growth factor.

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.

Structural organisations of hemoglobin and myoglobin influence their binding behaviour with phenothiazines

Binding modalities of chlorpromazine and trifluoperazine, two widely used antipsychotic phenothiazine drugs with hemoglobin and myoglobin have been studied to understand how the quaternary, tertiary and secondary structural organisations of the proteins regulate the binding process. NaCl-induced alteration in the quaternary structure of hemoglobin influences its binding modality with phenothiazines. Minor alterations in the tertiary structure of thermally denatured myoglobin (denaturation temperature ranging between 30–70°C) do not affect its affinity and the modality of binding with the drugs, but alterations in the secondary structure of the protein denatured at temperatures between 70–80°C influence its binding.

Copper(II) complexes with tridentate O,N,O-chelate dianions and additive monodentate donor ligands I. Crystal structures and EPR results of urea adducts of N-salicylidene- α-aminoacidato-copper(II) compared with N-salicylideneglycinato-thiourea-copper(II)

Dinuclear Cu(II) complexes with the tridentate N-salicylideneglycinate (saa 2−) or N-salicylidenemethylalaninate dianion (sab 2−) and urea (Ur) or thiourea (Tu) as additional monodentate donor ligand were prepared and characterised, namely [(saa)Cu(Ur)] 2 ( 1). [(sab)Cu(Ur)] 2(Ur) 2 ( 2) and [(saa)Cu(Tu)] 2 ( 3). The urea adducts crystallise in the triclinic space group P l with a = 7.611 ( 1), b=7.815(1), c=10.078(1) A ̊ ; α=98.06( 1), β=110.31(1), γ=95.91(1)° for 1 and a=8.820(1), b=9.712(1), c=10.317(1) A ̊ ; α=66.208(5), β=88.722(5), γ=83.364(6)° for 2; in each case Z=2 refering to the mononuclear constituents of centrosymmetric dimers. In the crystal structure of the thiourea derivative (monoclinic space group l2 a ; a=12.353(1), b=14.315(1), c=13.670(1) A ̊ ; β=91.62(1)°; Z=8), however, the dimers have C 2 symmetry due to a shift of the ligands into cis position. In all three cases the phenoxy-O ligands act as bridges in connecting monomeric units. Each one of them is part of the basal plane of one Cu ion and acts as an apical ligand to the copper centre of the second monomeric complex. The copper ions adopt (4 + 1) square-pyramidal geometry with relatively short apical CuO distances of about 2.4 Å in the case of 1 and 2 compared to about 2.8 Å for 3. Minor structural alterations in changing from 1 to 2 give rise to a significant impact in spectroscopic data, especially in the EPR parameters. The results are interpreted as consequences of (i) the electron inducing effect due to alkylation of the tridentate ligand and (ii) the intercalation of urea dimers in the case of 2 stabilised by hydrogen bonds.

Requirements for Epstein-Barr Nuclear Antigen 1 (EBNA1)-induced Permanganate Sensitivity of the Epstein-Barr Virus Latent Origin of DNA Replication

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin of DNA replication, oriP. EBNA1 binds cooperatively to four recognition sites in the dyad symmetry (DS) element of oriP, causing alterations in the origin DNA structure, which can be detected by the increased sensitivity of one Thy residue in two of the binding sites to permanganate oxidation. To better understand the significance of this EBNA1-induced origin distortion, we have investigated the DNA sequence and EBNA1 amino acid requirements for permanganate sensitivity. We have shown that the EBNA1 DNA binding and dimerization domains are sufficient to induce permanganate sensitivity and that amino acids 463-467, which form an extended chain that travels along the minor groove of the EBNA1 recognition site, play an important role in generating the DNA distortion. The EBNA1-induced permanganate sensitivity is independent of cooperative interactions between EBNA1 molecules on the origin and requires a specific sequence within the EBNA1 binding site. Using synthetic EBNA1 binding sites, we found that the inversion of a single AT base pair in the EBNA1 recognition sequence is sufficient to confer EBNA1-induced permanganate sensitivity. These studies indicate that permanganate oxidation can detect very minor alterations in DNA structure.

Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

Alterations to the highly conserved Asp549 of the retroviral ribonuclease H (RNase H) domain were evaluated in the heterodimeric (p66/p51) reverse transcriptases of human immunodeficiency and equine infectious anemia viruses. In addition to the polymerization-dependent and -independent modes of template hydrolysis, mutants were evaluated via their ability to select and extend the 3' polypurine tract (PPT) primers of these two lentiviruses into (+) strand DNA. Concerted and two-step reactions were designed to evaluate (+) strand priming, the latter of which allows discrimination between selection end extension events. In contrast to enzyme mutated at the highly conserved Glu478, substitution of Asp549 with Asn or Ala reduces, rather than completely eliminates, RNase H activity. When the requirement for RNase H function becomes more stringent, differences in activity are readily evident, most notably in the cleavage events liberating the 5' terminus of the PPT primer. PPT selection thus appears to represent a specialized form of RNase H activity that is more sensitive to minor structural alterations within this domain and may provide a novel therapeutic target.

Protease-resistant L-selectin mutants. Down-modulation by cross-linking but not cellular activation.

The adhesion molecule L-selectin (CD62L) is rapidly shed from the plasma membrane during leukocyte activation as a result of proteolytic cleavage between Lys321 and Ser322 within the extracellular domain. L-selectin is also down-modulated from the surface in response to cross-linking, possibly through a similar mechanism. To further characterize the mechanism of down-modulation, several L-selectin mutants were generated and transfected into COS cells. Wild-type L-selectin as well as mutants with one or two amino acid substitutions at the cleavage site were nearly quantitatively shed into the culture supernatant. However, mutants in which a nine-amino acid stretch that included the protease-sensitive site was either deleted or replaced with a polyglycine spacer or a comparable region of E-selectin were retained on the cell surface and not detected in the supernatant. These results are consistent with other reports describing protease resistant L-selectin mutants. We also demonstrate that when expressed in L1-2 pre-B cells, the L-selectin nine-amino acid deletion mutant (321del.9), but not wild-type L-selectin, is resistant to down-regulation induced by PMA. However, both wild-type and mutant 321del.9 are completely lost from the cell surface in response to cross-linking with an L-selectin Ab. PMA-induced- but not L-selectin cross-linking-induced down-modulation was inhibited by staurosporine. These data are consistent with the idea that the L-selectin protease(s) can tolerate minor structural alterations at the cleavage site, and that L-selectin down-modulation can be induced by more than one mechanism, at least one of which (cross-linking) is protein kinase C independent.

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.