I apologize that my rough draft is in point form.

I have arranged it this way because this format helps me break down the paper in to section and help organize my thoughts (so I can add/adjust information at ease). Sorry again! Introduction Answer questions: o o What is kariya ? when was it discovered Why are we studying it, why is it interesting

In 1982 an abnormal haemoglobin was detected in a healthy 19 yr old Japanese male It was fast moving, similar to Hb I (Lys -16 a replaced with Glu) Hb Kariya is a variant of Hb A with the Lys-40 a replaced with Glu By studying this variant using various analytical methods, one can see the functional role of the salt bridge between the e amino group of lys 40 and the COOH His group of the B chain In x-ray analysis, it was discovered that the a1b2 contact was located at the 40th position of the alpha chain In deoxy Hb A, Lys 40 forms salt bridge with COOH terminus of His (146 B), also the imidazole of the His forms intrachain with y COOH of Asp 94 B, these bridges stabilize the deoxy T state (low oxygen affinity) In T state, Tyr side chain adjacent to terminal His is in a pocket between helices E and H, when deoxy Hb -> oxy Hb, the pocket shrinks and the Tyr side chain is squeezed out This pulls the His away from the salt bridges formed with Lys 40a and Asp 94b, stabilizing the R state Also the imidazole undergo lower pka values and donates a proton which accounts for 40% of the Bohr effect Hb Kariya is interesting because it is the only known Hb which replaces Lys 40 with Glu This provides us with the opportunity to investigate how Lys-40 salt bridge with His 146 affects the function of Hb A and if there is a direct relationship between the interchain link and the stability of T-state

Methods and Procedures: Measured oxygen equilibrium curves under various conditions o Automatic oxygenation apparatus developed by imai polarographic (voltammetry measurement -> use electrodes) determination of partial pressure of oxygen (have an oxygen electrode) spectrophotometric determination of oxygen saturation of haemoglobin; machine used: Cary 118C Varian Associates Microcomputer model PDP-111 to measure data

Properties such as oxygen affinity, cooperativity of oxygen binding, bohr effect were all expressed as partial pressure of oxygen at half saturation point P50, Hill coefficient, n max, max slope of hill plot, and bohr coefficient

Measured reaction rate of sulfhydryl groups of cysteins-93(F9)b with 4-PDS (4-4 dipyridine disulphide) in oxy and deoxy forms o Spectrophorometer analysis described by Ampulski in alanlytical biochem journal Useful for measuring kinetics and amount of reactive sulfhydryl groups Hemoglobin has first order reaction rate

Measure OD (optical density) of standard and sample solution before addition of PDS Then in sample solution add PDS solution and start to record the change in optical density ( Computer will do this) Stop recording when the change in OD is less than 0.005 per minute Plot OD vs minutes graph

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Measure visible and soret region absorption spectra o Analyse methemoglobin content

Methemoglobin is when the iron of the heme group is ferric (3+) instead of ferrous (2+), cannot bind to oxygen, depends on methmoglobin reductase to convert back to hemoglobin Methemoglobin appears bluish chocolate brown color Measure content by double beam spectrophotometer (model 320L, Hitachi Co) at 560 nm, 576nm and 630 nm Measure immediately before and after oxygen equilibrium measurement

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Measure uv region derivative spectra and oxy minus deoxy difference spectra o o UV Derivative spectra used for qualitative analysis and quantification First order derivatives show the rate of change of absorbance with respect to wavelength, also has an inflection point at the wavelength of maximum absorbance Oxy minus deoxy difference spectra was measured using the same procedures as rate of sulfhydryl determination (ampulski)

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Resonance and Raman spectra of purified Hb Kariya and Hb A o Resonance raman scattering excited by 441.6 nm line of He/Cd laser, recorded with JEOL-400D Raman spectrometer RR spectorcopy provides info about vibrations of molecules, can be used to identify unknown substances

Discussion The Cys 93 b reactivity of SH group increased 43 fold in the deoxy state o o This is caused by the lack of interchain links of lys 40 a with His Usually bridges of lys 40 His and His-asp form a filter or a wall which prevent SH reagents from accessing the cys 93b

Peak height of soret spectra is reduced in deoxy Hb Kariya in comparison to deoxy Hb A o Peak height is reduced for deoxy Hb with high oxygen affinity and low cooperativity

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Shows that Hb Kariya is mainly in R state after deoxygenation

In UV derivative spectra, Hb A absorbance is halved upon deoxygenation at 290nm,Hb Kariya only decreased 30% upon deoxygenation o In UV derivative spectrum is mostly based on side chains Trp-37b and Tyr 42a, located at a1/b2 contact area In R state, Trp and Tyr are close therefore increase in absorbance, T state they are further apart We can conclude that in deoxy Hb Kariya mainly resides in R state

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All experimental data show that Tstate is destabilized in deoxy Hb Kariya After reading the paper, I learned that Protein function depends heavily on its structure This experiment proves that Lys-40 a salt bridge with His 164 b plays an important role in stabilizing the T-state By replacing Lys with Glu, there is no H bonds and electrostatic interactions in that position, reduces the strain of the protein, stabilizing the R-state These interactions are the directly related to the function, by changing one amino acid, the oxygen affinity of Hb Kariya increased 9x, Bohr effect decreased 30%, and coopertivity decreased as well