Photodegredation of Protein Light Harvesting Complexes In 4ul Microvolume Samples

Significance of the Topic

The development of microvolume UV/Vis/NIR spectrophotometry has revolutionized the analysis of scarce or highly concentrated biological samples. Measuring volumes as low as 0.8 µL with research-grade precision enables studies on proteins, nucleic acids or light-harvesting complexes previously limited by sample availability. This capability supports biochemical research, quality control in biotech applications and fundamental photochemical investigations.

Objectives and Study Overview

This study aimed to evaluate the performance of a Cary series spectrophotometer coupled with a Hellma TrayCell for measuring photobleaching effects in a 4 µL sample of bacterial light-harvesting complexes. Key goals included:

• Determining the absorbance profile in the UV/Vis range (200–800 nm).
• Assessing sample stability under repeated scanning to detect photodegradation.
• Demonstrating quantitation limits for low-concentration protein solutions.

Methodology and Instrumentation

Samples of purified bacterial porphyrin complexes were prepared at concentrations ranging from 20 µg/mL to 100 000 µg/mL. Spectral scans were conducted under these conditions:

• Wavelength range: 200–800 nm
• Bandwidth: 2 nm
• Scan speed: 60 nm/min
• Averaging time: 2 s, data interval: 2 nm
• Rear beam attenuation set to offset TrayCell internal attenuation (1.3 Abs)
• TrayCell transmission aligned to 4.8 %T

Instrumentation Used

• Agilent (formerly Varian) Cary 6000i UV/Vis/NIR spectrophotometer
• Hellma TrayCell microvolume sample holder with adjustable path length (0.2–1 mm)
• Automated and manual rear beam attenuators

Main Results and Discussion

Repeated scans at 400 nm revealed a progressive decrease in absorbance, indicating photobleaching at peak wavelengths. In contrast, scans at 500 nm showed stable absorbance (variance <0.0008 Abs), confirming minimal baseline drift. The noise level of 0.0002 Abs in 1 µL samples enabled detection down to approximately 100 µg/mL protein or 4 ng DNA. The increase in optical path length from 0.2 mm to 1 mm further improved sensitivity for samples as dilute as 20 µg/mL.

Benefits and Practical Applications

The microvolume approach offers significant advantages:

• Minimal sample consumption preserves precious biomolecules.
• High dynamic range (up to 7 Abs in TrayCell) supports broad concentration measurements.
• Rapid purity assessment via full spectral scans enhances QA/QC workflows.

Future Trends and Possibilities

Advancements may include integration with microfluidic sampling, real-time monitoring of photochemical reactions and automated photostability screening for drug development. Combining microvolume spectrophotometry with advanced data analytics could further optimize low-volume assays in proteomics and genomics.

Conclusion

This work demonstrates that microvolume UV/Vis/NIR spectrophotometry on Cary instruments with a Hellma TrayCell allows precise quantitation and photostability analysis of protein complexes in volumes as low as 4 µL. The approach opens new avenues for studying fragile or limited samples with minimal material consumption.

Reference

• Hunter CN, Department of Molecular Biology and Biochemistry, University of Sheffield – purification of bacterial light-harvesting complexes.