Oscar H. Kapp

Structure and function of invertebrate oxygen carriers

Oxygen binding proteins are large multi-unit proteins ideally suited to the study of structure function relationships in biological molecules. This book, based on a Symposium at the 10th International Biophysics Congress in 1990, provides a synthesis of recent advances in our knowledge of invertebrate oxygen carriers such as haemoglobins, haemocyanins and haemorythrins. Reviews of these are combined with new research results of interest to biochemists and molecular biologists concerned with oxygen carriers in general, their gene structure and comparative biochemistry. Of particular value are the studies of invertebrate oxygen binding proteins which perform their function and have structures vastly different from the vertebrate haemoglobins and myoglobins, as well as examples of modern molecular techniques. This book of proceedings on comparative physiology, biochemistry and physical biochemistry is intended for libraries and researchers.

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

The molecular size of Myxicola infundibulum chlorocruorin and its subunits

The molecular shape and size of the extracellular chlorocruorin of Myxicola infundibulum was determined using scanning transmission electron microscopy and its dissociation in the presence of sodium dodecyl sulfate (SDS) was investigated using polyacrylamide gel electrophoresis. The shape of the chlorocruorin is that of a two-tiered hexagon with a vertex-to-vertex diameter of 29.0-29.5 nm and a height of 19.0-19.7 nm: it appears to be smaller by 5-10% relative to several annelid extracellular hemoglobins examined by scanning transmission electron microscopy. The quaternary structure of the chlorocruorin appears to be sensitive to Ca(II) concentration; dissociation fragments of the whole molecule were observed, consisting of octamers an dimers of one-twelfth subunits. The unreduced chlorocruorin dissociated into two subunits with estimated molecular masses of 23 000 (1) and 60 000 (2); the reduced chlorocruorin dissociated into subunits with estimated molecular masses of 13 000 (I), 14 000 (II) and 30 000 (III). SDS-polyacrylamide gel electrophoresis of reduced subunits 1 and 2 showed that subunit 1 corresponded to subunit III and that subunit 2 dissociated to subunits I and II. Densitometry of the polyacrylamide gels indicates that 85-90% of the Myxicola chlorocruorin consists of disulfide-bonded tetramers of polypeptide chains of about 15 000. Such a pattern of subunit aggregation has not been observed previously in annelid extracellular hemoglobins and chlorocruorins.

Modification of a scanning electron microscope to produce Smith–Purcell radiation

We have modified a scanning electron microscope (SEM) in an attempt to produce a miniature free electron laser that can produce radiation in the far infrared region, which is difficult to obtain otherwise. This device is similar to the instrument studied by the Dartmouth group and functions on the basic principles first described by Smith and Purcell. The electron beam of the SEM is passed over a metal grating and should be capable of producing photons either in the spontaneous emission regime or in the superradiance regime if the electron beam is sufficiently bright. The instrument is capable of being continuously tuned by virtue of the period of the metal grating and the choice of accelerating voltage. The emitted Smith–Purcell photons exit the instrument via a polyethylene window and are detected by an infrared bolometer. Although we have obtained power levels exceeding nanowatts in the spontaneous emission regime, we have thus far not been able to detect a clear example of superradiance.

Comparison of the molecular size and shape of the extracellular hemoglobins of Tubifex tubifex and Lumbricus terrestris

Abstract The extracellular hemoglobins of Tubifex tubifex and Lumbricus terrestris have been examined by gel filtration and scanning transmission electron microscopy and a detailed comparison of their morphologies has been performed using digital image processing techniques. The hemoglobins possess identical elution volumes on analytical columns of Sepharose CL-6B and CL-2B. In electron micrographs the overall shape of negatively stained Tubifex and Lumbricus hemoglobins is similar to the two-tiered hexagonal appearance of other annelid extracellular hemoglobins. Tubifex hemoglobin possesses a vertex to vertex diameter of 31.3 nm and a height of 21.1 nm, while the corresponding dimensions of Lumbricus hemoglobin are 31.2 nm and 21.5 nm, respectively. These dimensions are about 25% larger than those obtained by conventional transmission electron microscopy but are consistent with other values obtained by scanning transmission EM (Kapp, O.H., Vinogradov, S.N., Ohtsuki, M. and Crewe, A.V. (1982) Biochim. Biophys. Acta 704, 546–548) and by X-ray diffraction of wet crystals of Lumbricus hemoglobin (Royer, W.E., Broden B.C., Jacobs, H.C. and Loves, W.E. (1981) in Invertebrate Oxygen-Binding Proteins (Lamy, J. and Lamy, J., eds.), 337–341, Marcel Dekker, New York). Comparison of the morphologies of the two hemoglobins by examination of individual pixel lines through their digital projections suggests that the molecules are very similar in overall structure.

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated Mm of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem.10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.

Calculation of subunit stoichiometry of large, multisubunit proteins from amino acid compositions

The subunit stoichiometry of a large, multisubunit protein can be determined from the molar amino acid compositions (i amino acids) of the protein and its subunits. The number of copies of the subunits (1, 2, ... j) is calculated by solving all possible combinations of simultaneous equations in j unknowns (i!/j!(i - j)!). Calculations carried out using the published amino acid compositions determined by analysis and the compositions calculated from the sequences for two proteins of known stoichiometry provided the following results: Escherichia coli aspartate transcarbamoylase (R6C6, Mr = 307.5 kDa), R = 5.6 to 6.6 and C = 5.8 to 6.3, and spinach ribulose-bisphosphate carboxylase (L8S8, Mr = 535 kDa), L = 7.3 to 9.1 and S = 5.6 to 10.6. Calculations were also carried out with the amino acid compositions of two much larger proteins, the E. coli pyruvate dehydrogenase complex, Mr = 5280 kDa, subunits E1 (99.5 kDa), E2 (66 kDa), and E3 (50.6 kDa), and the extracellular hemoglobin of Lumbricus terrestris, Mr = 3760 kDa, subunits M (17 kDa), D1 (31 kDa), D2 (37 kDa), and T (51 kDa); the results for PDHase were E1 = 20 to 24, E2 = 18 to 31, E3 = 21 to 33 and those for Lumbricus hemoglobin were M = 34 to 46, D1 = 13 to 19, D2 = 13 to 18, and T = 34 to 36. Although the sample standard deviations of the mean values are generally high, the proposed method works surprisingly well for the two smaller proteins and provides physically reasonable results for the two larger proteins.

An image acquisition system built with a modular frame grabber for scanning electron microscopes

We have built an image acquisition and processing system based on a modular frame grabber board (MFG) for use with scanning (or scanning transmission) electron microscopes. The variable‐scan acquisition module of the grabber board provides compatibility with electron microscopes processing various scan speeds, e.g., the very slow scan rate of our mirror‐type electron microscope. In addition to the acquisition function, the board provides many image processing capabilities. A special time‐base unit was built to synchronize the acquisition system with the scanning system on the electron microscope. A Windows application has been built to operate the MFG as well as manage all functions of the electron microscope. Using this approach we have been able to greatly simplify the task of digital image acquisition as well as creating a powerful and seamless interface to our Windows‐based environment.

Integrated windows‐based control system for an electron microscope

A Windows application has been developed for management and operation of beam instruments such as electron or ion microscopes. It provides a facility that allows an operator to manage a complicated instrument with minimal effort, primarily under mouse control. The hardware control components used on similar instruments (e.g., the scanning transmission electron microscopes in our lab), such as toggles, buttons, and potentiometers for adjustments on various scales, are all replaced by the controls of the Windows application and are addressable on a single screen. The new controls in this program (via adjustable software settings) offer speed of response and smooth operation providing tailored control of various instrument parameters. Along with the controls offering single parameter adjustment, a two‐dimensional control was developed that allows two parameters to be coupled and addressed simultaneously. This capability provides convenience for such tasks as ‘‘finding the beam’’ and directing it to a locatio...