Laser Raman spectra of calf thymus chromatin and its constituents

Savoie, Rodrigue; Jutier, Jean-Jacques; Alex, Serge; Nadeau, Paul; Lewis, Peter N.

doi:10.1016/s0006-3495(85)83937-0

Cited by 20 publications

(8 citation statements)

References 43 publications

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“…It is clear therefore that the proteins in the nucleus possess predominantly ahelix and random coil domains. These findings are in accordance with Raman spectroscopic data obtained from isolated nucleosomes, nucleosome cores, and from isolated Calf Thymus and chicken erythrocyte chromatin containing little or no nonhistone proteins (NHP) (26)(27)(28). As demonstrated below NHP is abundantly present in the nuclei of granulocytes.…”

Section: -Sheet Signal Contributions Would Have Been Expectedsupporting

confidence: 90%

“…The intensity of 1,094 cm-' DNA line is virtually insensitive to DNA-protein interactions in nucleosomes and chromatin (26)(27)(28) and in DNA-polylysine and DNA-polyarginine complexes (29,30). The intensity of 1,449 cm-' protein line is insensitive to protein secondary structure and depends only on the number of protein CH2 and CH3 groups (2,40).…”

Section: Determination Of Dna-protein Ratiomentioning

confidence: 99%

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Raman microspectroscopic approach to the study of human granulocytes

Puppels

Garritsen

Segers-Nolten

et al. 1991

Biophysical Journal

170

141

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A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

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Section: -Sheet Signal Contributions Would Have Been Expectedsupporting

confidence: 90%

Section: Determination Of Dna-protein Ratiomentioning

confidence: 99%

Raman microspectroscopic approach to the study of human granulocytes

Puppels

Garritsen

Segers-Nolten

et al. 1991

Biophysical Journal

170

141

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“…However, since no measurements were carried out at pH's lower than 1.8, this will not affect the general validity of the results presented here. The intensity of the 1094-cnr1 line is furthermore largely independent of the DNA-protein interactions that occur in nucleosomes and chromatin (Thomas et al, 1977;Hayashi et al, 1986;Savoie et al, 1985) and in DNA-polylysine complexes (Prescott et al, 1976).…”

Section: Methodsmentioning

confidence: 99%

“…Alterations in molecular structure lead to changes in the Raman spectrum. The technique has been widely applied to study the composition, structure, and interactions of nucleic acids and proteins (see, e.g., Erfurth & Peticolas, 1975; Goodwin & Brahms, 1978;Hayashi et al, 1986;Savoie et al, 1985;Thomas et al, 1977Thomas et al, , 1986. Measurements were made of chromosomes at low pH, chromosomes reneutralized after a brief incubation at low pH, and calf thymus DNA at low pH.…”

mentioning

confidence: 99%

Raman Microspectroscopic Study of Low-pH-Induced Changes in DNA Structure of Polytene Chromosomes

Puppels¹,

Otto²,

Greve³

et al. 1994

Biochemistry

137

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The effects of low-pH treatments on DNA structure in polytene chromosomes of Chironomus thummi thummi have been studied by Raman microspectroscopy. Measurements were carried out on chromosomes at low pH and on chromosomes reneutralized after a short exposure to low pH. Protonation of adenine residues and subsequent unpairing of adenine (A) and thymine (T) were found to commence already above pH 3.6 and are completed at pH 2.2. Protonation of the cytosine-guanine base pair also starts above pH 3.6. It leads to an intermediate protonated, most likely Hoogsteen-type, guanine-cytosine base pair. Unpairing of G and C residues starts between pH 2.6 and 2.2 and continues below pH 2.2. Brief treatment of chromosomes at a pH < or = 2.2, i.e., at a pH where both AT and GC base pairs are disrupted, was found to lead to irreversible changes in DNA conformation upon return to neutral pH. These were most pronounced after treatment with 45% acetic acid. More than 10% of the A and T residues was found to have adopted a non-B-DNA conformation. Evidence was found for denaturation of the B-form backbone. The amount of protein extracted from the chromosomes was strongly pH-dependent. Treatment at pH 3.6 did not cause noticeable protein extraction, while treatment with 45% acetic acid extracted more than 50% (by weight) of the chromosomal proteins.

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“…An experiment (not shown) in which a mixture of glucose oxidase and catalase was added to a metaphase chromosome preparation showed that damage still occured. Such an enzyme mixture was shown [24] to be very effective in depleting a sample of free oxygen molecules. However, reactive hydrogen peroxide may be present and the enzymes contain tlavines which can act as sensitizers [23].…”

mentioning

confidence: 99%