M.W. Pandit

Studies on silk fibroin. I. Molecular weight, sedimentation coefficient, viscosity and optical rotation of silk fibroin from carbonate-extracted silk fiber.

Abstract 1. 1. The molecular weight, sedimentation coefficient, intrinsic viscosity and optical rotation in the visible region of silk fibroin obtained by extraction of silk fiber with boiling Na 2 CO 3 solution for different intervals of time were measured. The preparations obtained by extraction for 30, 60, 90, 120, 150 and 180 min were studied. 2. 2. The preparations were heterogeneous as judged by a decrease in molecular weight with the period of centrifugation. The heterogeneity decreased as the period of Na 2 CO 3 extraction increased. The true weight-average molecular weight of each sample was obtained by extrapolation of the data to t = o of centrifugation and these values also decreased with the period of extraction. 3. 3. Extraction of silk fiber with boiling Na 2 CO 3 solution caused degradation of fibroin. Autoclaving of silk fiber in distilled water also caused degradation of fibroin. 4. 4. Silk fiber dissolved to the extent of nearly 80% in neutral or acidic (pH 4) saturated LiCNS solution and dissolved completely in alkaline (pH 10) saturated LiCNS solution. The molecular weight of the protein in neutral solution was 1.8 × 10 6 , in acidic solution 3.0 × 10 5 and in alkaline solution 2.0 × 10 3 . 5. 5. The sodium-carbonate extracted fibroin samples gave a single peak in sedimentation velocity experiments and exhibited different concentration-dependence of S 20,w . The extrapolated S 20,w value decreased as the period of Na 2 CO 3 extraction increased. 6. 6. The intrinsic viscosity also decreased with the period of extraction and the viscosity values were considerably higher than those obtained for native globular proteins. 7. 7. An analysis of the optical rotation data with the Moffitt equation showed that the fibroins had very low helical content. 8. 8. The plot of the logarithm of intrinsic viscosity or the logarithm of sedimentation coefficient against the logarithm of molecular weight was linear suggesting that the fibroin preparations behaved as random-coiled polypeptides.

Denaturations studies on bovine pancreatic ribonuclease: Effect of trichloroacetic acid

Exposure of ribonuclease (EC 3.1.27.5) to 5% trichloroacetic acid solution is found to partially inactivate the enzyme. This inactivation is a function of time of exposure to trichloroacetic acid and reaches a plateau of about 45% residual activity. Higher concentrations of trichloroacetic acid lead to greater inactivation. Physicochemical properties such as sedimentation coefficient, gel-filtration behaviour and polyacrylamide gel electrophoresis of the trichloroacetic acid-treated enzyme remain unaffected as compared to the untreated enzyme. However, spectrophotometric titration of the trichloroacetic acid-treated enzyme revealed that one of the three buried groups of tyrosine is exposed to the outside surface of the molecule. Near ultraviolet CD spectra supported these observations. Far ultraviolet CD spectra suggested some refolding of the enzyme after trichloroacetic acid treatment. Immunological determinants on the molecule remain unaltered upon trichloroacetic acid treatment. It is concluded that the exposed tyrosine group may be causing a conformational change in the protein and this change may be indirectly responsible for the observed reduction in the activity after trichloroacetic acid treatment.

The oxalic acid catalysed oxidation of bis(2,4,6-tripyridyl-1,3,5-triazine)-iron(II) by chromium(VI) in acetate buffer

SummaryThe reaction between bis(2,4,6-tripyridyl-1,3,5-triazine)-iron(II), Fe(TPTZ)inf2sup2+and chromium(VI) in acetate buffers is very slow. However, in the presence of oxalic acid (catalyst) it is very fast and is completed within 10s. The reaction was studied in the 3.6–5.6 pH range using stopped-flow spectrophotometry. The reaction is first order in the substrate and zero order in the oxidant. The rate of the reaction increases with the increase in pH. Kinetic evidence for complexation between the substrate and the catalyst was obtained and a mechanism involving the formation of an ion-pair between Fe(TPTZ)inf2sup2+and the oxalate ion is proposed.

Helix-destabilization of deoxyribonucleic acid and poly[d(A-T)·d(A-T)] by bovine seminal ribonuclease

A ribonuclease isolated earlier from bovine seminal plasma by DNA-affinity chromatography (Ramakrishnamurti, T. and Pandit, M.W. (1983) J. Chromatogr. 260, 216-222) has now been shown by thermal denaturation studies to destabilize the double-helical structure of DNA and poly[d(A-T).d(A-T)]. Thermal denaturation profiles of DNA in the presence of the protein are much more complicated due to the denaturation of protein itself in the temperature range over which DNA predominantly melts. The protein shows relatively stronger affinity towards denatured DNA as compared to native DNA. The action of micrococcal nuclease on DNA and its complexes with ribonuclease A and bovine seminal ribonuclease indicates that both of these proteins destabilize the double-helical structure of native DNA and thereby render the DNA more sensitive to the micrococcal nuclease.

Kinetics and mechanism of the oxidation ofbis(2,4,6-tripyridyl-1,3,5-triazine)iron(II) bytrans-1,2-diaminocyclohexanetetraacetatomanganate(III) in acetate buffer

SummaryThe rapid oxidation ofbis(2,4,6-tripyridyl-1,3,5-triazine)-iron(II), [Fe(TPTZ)2]2+, bytrans-1,2-diaminocyclohexanetetraacetatomanganate(III), [MnIII(Y)]−, in acetate buffers was monitored using stopped-flow spectrophotometry. The reaction is first order in the substrate and evidence was obtained for pre-complexation between the oxidant and the substrate. The reaction rate increases as the pH increases. Characterisation of the products using the radiotracers54Mn and59Fe indicated that [MnII(Y)]2− and [Fe(TPTZ)2]3+ are the final products. The reaction obeys the rate law:n

Self-association of diisopropylphosphoryl-α-chymotrypsin: the involvement of active site of the enzyme in its self-association behaviour

k_{obs} = frac{{k_3 K_2 [Mn^{III} (Y)^ - ]_t }}{{{ 1 + K_1 [H^ + ] + K_2 [Mn^{III} (Y)^ - ]_t } }}

Identity of the ribonuclease from bovine seminal plasma with ribonuclease BS-1, and its sensitivity to polyvinyl sulphate.

n

Rapid purification of a ribonuclease from bovine seminal plasma by DNA-affinity chromatography.

Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2:3-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.